RO128710A0 - Phytotherapeutic products with cumulated effect in cutaneous inflammatory disorders - Google Patents

Abstract

The invention relates to a phytotherapeutic product used in dermatology. According to the invention, the product comprises 0.1...12% extract of herba Trifolii, possibly extract of Hippocastani, and 1...2% extract of folium Hederae, extract of radix Bardanae, 0.1...2.5% extract of herba Cyani, and usual conditioning agents for the remainder up to 100%, the percentage being expressed by weight.

Description

The present invention relates to herbal products with cumulative action of dermal and anti-inflammatory restructuring used in the treatment of different pathophysiological manifestations of skin tissue: cellulite, peripheral edema, ointment.

The patent proposes the association of the active principles isolated from different plants, in order to cumulate the dermal restructuring actions by stimulating the cell and protein tumors as well as the cell adhesion - extracellular matrix, photoprotective through cell protection against the induction of apoptosis, antioxidant effect, anti- inflammatory and anti-angiogenic against UV radiation, anti-inflammatory at vascular level by inhibition of pro-inflammatory cytokines IL6 and IL8 and overexpression of monocyte - endothelial adhesion molecules (ICAM and VCAM) and vascular antipermeabilization by VEGF factor inhibition, evidenced by tests specific for each active cosmetic product, namely:

- selective extract called Dermo ET, isolated from Grass Trifolii, conditioned in the form of fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in flavonoid compounds expressed in quercetin of min.0,15 g / 100 ml and in isoflavonic aglycones of min. 0.23 g / 100 mL as the sum of daidzein, genistein, formononetin and biochanin A, quantitatively determined by HPLC, namely: daidzeine min. 10 mg / 100 mL, genistein min. 20 mg / 100 mL, formononetin min. 140 mg / 100 mL and biochanin A min. 60 mg / 100 mL, obtained in ratio 2: 1-1: 1 plant: final extract;

- selective extract called Dermo Cas, isolated from Hippocastani semen, conditioned either in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in proanthocyanins of min. 0.15 g / 100 g and total triterpenic saponins expressed in min. 2 g / 100 g, obtained in a ratio of 1: 1 plant: final extract; or in the form of a dry extract in the form of a non-hygroscopic yellow-brown powder, called Dermo Es, standardized in triterpenic saponins expressed in a min.70% scale, and in flavonoids of min 2% obtained in a ratio of 33: 1 plant: final extract;

- selective extract called Dermo EH, isolated from Folium Hederae Helicis, conditioned either in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0,3 g / lOOg, and in saponins total triterpenes expressed in hederacozide C of min.3 g / 100 g, obtained in the ratio 1: 1-1: 2 plant: final extract, or in the form of dry extract in the form of a white-brown, nonhygroscopic powder,

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^ -2012-00618-1 3 -08- 2012 named Dermo HdC standardized in triterpenic saponins expressed in C hederacozide min. 60%, obtained in 20: 1 ratio plant: final extract;

- selective extract called Dermo B, isolated from Radix Bardanae, conditioned in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in polyphenol carboxylic acids expressed in caffeic acid by min. 0,3 g / 100 g,, obtained in a ratio of 1: 1 plant: final extract;

- selective extract called Dermo Abs, isolated from Cyani Grass, conditioned as a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0,9 g / 100 g and in flavonoid compounds expressed in the routine of min.0,5 g / 100 g, obtained in the ratio 1: 1-1: 2 plant: final extract;

The patent presents the complementary and synergistic biological effects of the active cosmetic products that highlight the dermal, anti-inflammatory and anti-dermal restructuring properties at the skin level, but also the conditioning variants of the active components, in the form of cosmetic products, developed based on the combination of the target actions demonstrated at cellular level.

According to the present patent, obtaining Trifolium pratense crops used as plant raw materials for obtaining selective Dermo ET extract is carried out in ecological system. Organic agricultural crops represent the solution to solve the problems related to satisfying the increasing demand for organic natural products, which do not involve the use of synthetic chemicals. The lack of pesticides remaining in the plant used as a source of raw materials is a desire of the modern pharmaceutical and cosmetic industry, especially due to the therapeutic uses of phytocompounds. In the organic cultivation technology of the red clover, the methods of preparing the land, setting up and maintaining it, combating diseases and pests, as well as harvesting have been carried out in accordance with the principles of organic farming, being novelty for our country. Another innovative aspect is the use in the ecological culture of the variety Pratorum, cultivated without a protective plant at a distance between 25 cm rows.

In the case of the sources of plant raw materials - Hippocastani semen and Folium Hederae Helicis, they come from ecologically protected areas, and Herba Cyani and Radix Bardanae are obtained from batches of experimental testing in order to approve ecological culture technologies. The novelty character is the introduction into the culture (the test batch) of the two species of plants (the blue and the combs) and obtaining for the first time in our country raw materials, ecologically from surfaces cultivated ecologically, not from spontaneous flora.

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Technologies for extracting extracts! sc.c; .?. 'e empLcU. i: carrying out the inventions involves minimum energy consumption and optimum yields, not maximum, using as far as possible as extraction means substances of natural origin: water, ethyl alcohol, glycerin, vegetable oils.

The technology at the industrial level for obtaining therapeutic and / or cosmetically active components, proposed according to the invention, has several advantages:

- the extraction plant is simple but very efficient being made of extraction vessels and stainless steel tanks - for solvent and extractive solutions - between which a solvent / mixture recirculation loop was mounted, in which 2-3 centrifugal pumps are located whose speed of 2900 rpm print a large capacity of extraction of the components of the plant;

- the extraction of components uses as far as possible as extraction means substances of natural origin: water, ethyl alcohol, glycerin, according to the principles of ecological technologies;

- being environmentally friendly technologies, avoids high processing temperatures, which means reduced energy consumption;

- the extraction stages are carried out at ambient temperature;

- the whole process avoids extreme pH values, usually taking place at the usual values of solvents;

- the extraction time is much reduced due to the high recirculation speed of the solvent / mixture;

- the concentration stages are performed under vacuum, thus increasing the distillation capacity and reducing the time of operations;

- the active components obtained representing cosmetic ingredients, are conditioned in different cosmetic skin care products of anti-cellulite, anti-oily, anti-dandruff type; their effectiveness is based on the cumulative, synergistic action demonstrated by tests at the target cell level, confirmed and by clinical studies on human volunteers.

Most pathophysiological processes in the body are a combination of triggering factors and complex biological processes that underlie their installation.

Thus, cellulite is a dermatocosmetic disorder with a high degree of complexity, involving inflammatory processes in the circulatory and lymphatic system, structural changes of the extracellular matrix, excess subcutaneous adipose tissue due to disturbances of lipid metabolism (increased lipogenesis and slowing of lipolysis). [1-2].

Therefore, in the effective treatment of this condition it must be intervened both by reducing adipogenesis and by stopping vascular inflammation and restructuring the dermis affected by adipocyte insertions. [3]

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Another inflammatory process that is taking the border between therapy and achieving a comfortable comfort! the dermo-cosmetic care products it is the sensation of "tired legs" ¹ or "heavy, one of the first symptoms of peripheral vascular diseases. [4]

Of these, chronic venous insufficiency affects almost 40% of the population of developed countries. In the progression of this pathology there is an increase in the blood microcirculatory flow that causes the dilation of the capillaries, inducing venous hypertension, but also vascular permeability disorders, propagation of inflammation through the extracellular cytokine cascade, stimulation of adhesion molecules, but also dermal dermal changes. with supporting role for microvasculature. [5,6]

Thus, the optimal route of intervention is the concerted action at both blood vessel level, inflammatory endothelial process and dermal restructuring through inhibition of matrix metalloproteinases (eg MMP 9 is involved in venous tissue remodeling) and acceleration of protein biosynthesis.

Circumcision or periorbital hypereromy is another physiopathological manifestation of inflammatory type, mainly caused by dermal melanization and / or post-inflammatory hemodynamic congestion. [7]

However, histological studies have shown a weak correlation between the improvement of the cirrhosis and the reduction of melanosis, which suggests a different therapeutic intervention pathway than the modulation of the melanin quantity. There are environmental and congenital factors that converge on the appearance of the cecarines, including: UV radiation, chronological aging, psychic and emotional stress, allergic and atopic reactions, even estrogenic imbalance, all of which are convergent to the release of mediators of inflammation (cytokines, factors signaling proteins) that affect vascular permeability. [8,9]

The anti-inflammatory agents commonly used in dermatological products are steroid compounds such as corticosteroids such as: dexamethasone, hydrocortisone, triamycinolone alone or in combination, but due to the adverse systemic effects they cause, they are increasingly avoided. Also useful for the anti-inammatory effect are compounds that include non-steroidal substances of the type: piroxicam, tenoxicam, diclofenac, indomethacin, phenamate, ibuprofen, naproxen, ketoprofen, phenylbutazone, all compounds obtained by chemical synthesis.

The current trend is the replacement of synthetic therapeutic agents with natural ones, obtained from the plant world, of microorganisms or animals, which, although they usually have a lower pharmacological activity, are more efficient for long-term use, due to their safety in administration. usually devoid of major adverse reactions.

The official documents at European level lately [10] impose new regulations regarding the use of only natural ingredients, obtained by simple ecological methods / procedures,

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3 -08- 201Ζ it is forbidden to use substances dc arc coloration or antioxidant effect, obtained by chemical synthesis.

Thus, in the last decade, patents have emerged that claim the use of preparations of plant origin with different therapeutic effects: anti-inflammatory, antioxidant, for increasing the vascular permeability, lipolipemic agents e.t.c. So:

- US patent 7,098,189 / 29.08.2006 uses as natural anti-inflammatory various extracts obtained from: Candelilla wax, bisabolol, Aloe vera extract, various plant sterols, Rubia extracts, Commiphora, kola, chamomile, clover, Glycyrrhiza , glycyrrhetinic acid, but also its derivatives in the form of salts or esters. The concentrations of the active components are not specified, but only the proportion of them in the final cosmetic products.

- Patent application WO 1984/002846 proposes an ointment based on: enzyme-papain, bromelain, trypsin, chymotrypsin, pancreatin, lipase, amylase-, an aloe extract and an astringent skin treatment agent.

- EP patent 1932517 / 18.06.2008 for the treatment of cancer and inflammatory autoimmune syndromes proposes a liposomal composition containing polyphenol derivatives- mainly caffeic acid-, ferulic acid, luteolin, diosmin, hesperetin, naringenin, quercetin, catroline, resin . The sources of these compounds are not specified except in the case of caffeic acid.

Based on the idea that the commonly used anti-cellulite agents are xanthine compounds - caffeine, theophylline, theobromine and aminophylline [11], the present invention proposes associations between one of these, caffeine, known as fat-lipolysis accelerator and adipose tissue lipolysis. the bioactive principles obtained - by their own technologies - from the following plant species: Herb Trifolii, Hippocastani semen, Folium Hederae Helicis, with anti-inflammatory and dermo-restitutive effects. According to the invention, the active principles existing in the phytotherapeutic preparations and which are responsible for the cumulative effect, synergy, belong to the triterpenic saponins - escina and hederacozide C, polyphenols - caffeic acid, flavonoids, proanthocyanins, isoflavones and polyacetylene.

For the first stages of evolution of peripheral vascular edema, concerted action at the level of blood vessel and skin tissue refers to dermal restructuring by inhibition of matrix metalloproteinases and acceleration of protein biosynthesis, associated with stopping the vascular inflammatory process as a whole, or including vascular permeabilization through VEGF inhibition. This therapeutic target is achieved, according to the present invention, by combining the bioproducts from the ivy, red clover and brust.

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Also, based on the mechanisms of occurrence of orbital hyperchromia, a product was designed to combat this condition by combining bioactive components from the following plant species: Centaurea cyanus, Trifolium pratense, Aesculus hypocastanum.

Clover extract - Dermo-ET, by complementary action of the active ingredients (isoflavones: genistein, daidzein, biochanin, formononetin, and flavonoids of quercetin type, which justifies the antioxidant effect highlighted) has a dermal restructuring effect through 2 mechanisms: stimulation of collagen synthesis and inhibition of MMP9 (reconsolidation of the extracellular matrix with supporting role) and acceleration of cell proliferation of fibroblasts, induces the overexpression of integrins responsible for inter-cell adhesion and consequently the tightness of the skin tissue, coupled with the antioxidant and photoprotective effect against UV-A and IJV-B radiation. , including stopping angiogenesis. This is the component responsible for the dermal restructuring, ensuring the integrity of the structural support of the blood network, necessary in combating all 3 types of skin inflammatory disorders that are the subject of this patent.

The dry extract of wild chestnut seeds with a content of min.70% triterpenic saponins expressed in escina, as well as the extract of ivy leaves with a content of min.60% in triterpenic saponins expressed in hederacozide C, has a cumulative vascular anti-inflammatory effect, inhibiting the expression of ICAM and VCAM adhesion molecules and the secretion of pro-inflammatory cytokines IL6 and IL8 and also reduces vascular permeability (decreases VEGF factor release), in the sense of reducing edema. The combination of the two extracts with anti-inflammatory action is justified by their complementary effects, as follows: the dry extract of wild chestnut seeds inhibits angiogenesis produced by UV radiation - it stops the superficial vascularization of the skin, and the extract from the ivy leaves reduces the edema by decreased vascular permeability.

The dry ivy-Dermo EH extract inhibits the expression of ICAM and VCAM adhesion molecules and the secretion of pro-inflammatory cytokines IL6 and IL8 and reduces vascular permeability (decreases VEGF release), in the sense of diminishing edema. Its anti-inflammatory action is accentuated by that of the Dermo B burn extract which, besides stopping the vascular inflammation, also has antioxidant and antimicrobial effect.

Blue extract - Dermo Abs, as well as chestnut Dermo Cas - shows anti-inflammatory action at the vascular endothelium level (inhibition of ICAM adhesion molecule expression, and release of pro-inflammatory cytokines IL6 and IL8), by combining the two extracts obtaining has a potentiated effect. In addition, the components of the blue extract confer

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it has an antioxidant and antimicrobial effect, as well as anti-irritant against UV radiation (it inhibits IL-pro-irritant signaling molecule), important aspects in formulations for anatomically sensitive areas, photo-exposed. The complementarity of the action of the chestnut extract with the blue one is also manifested by the effect of stopping the angiogenesis generated by the UV radiation, this cumulation of effects conferring maximum efficiency in the sun protection.

At the global level, phytochemical and pharmacological researches are carried out intensively in order to obtain bioactive substances and products with therapeutic activity that are free of adverse effects and that can be used in dermatocosmetic or cosmetic products.

Flavones and isoflavones

Isoflavones, substances belonging to the class of polyphenols, can be obtained by synthesis or from plant sources, from plants belonging to Legume legumes. The role of isoflavones in plants seems to be hormones, but also has antioxidant and antibacterial properties. The most studied isoflavones are genistein and daidzein isolated from soy.

There are studies demonstrating the moderate estrogenic properties of genistein and daidzein isoflavones, administered as nutritional supplements, but the daily dose limitation due to insufficient safety data at 80 mg / day appears to be too low to show efficacy.

After ingestion of genistein in the form of supplements [12], it was determined the decrease of body mass and of the fat in the tissues, accompanied by the decrease of the appetite; also, alterations of the concentrations of hormones were recorded: insulin, thyroid, adenocorticotropic, cortisosteron and corticosteron. In addition, genistein content is associated with impaired expression of genes involved in lipid metabolism, and glucose transport is deficient in cells affected by lipolysis, lipogenesis, altering ATP synthesis.

Isoflavones - including soybean genistein [13] have antioxidant effects and protect cells against free radicals - inhibiting the expression of stress response, of the genes involved, thereby reducing carcinogenesis.

In PUVA therapy, genistein protects the skin from the effect of psoralens and UV radiation, which is associated with an increased risk of carcinomas and melanomas. [14]

Miyazaki K et al. [15] studied the effect of daidzein and genistein on hyaluronic acid on cell cultures of human keratinocytes and on the skin of mice epilated by topical applications for 2 weeks. The result showed a significant increase in the production of hyaluronic acid / both in vitro and in vivo experiment. It is thus suggested that both genistein and cat

and daidzein through topical applications prevent and improve skin alterations caused by the loss of hyaluronic acid in the skin.

The benefits that can derive from topical application of cosmetic compositions seem to be superior to those that can be obtained by systemic ingestion of these active substances, for the following reasons:

- The bioavailability of isoflavones in the skin appears to be higher than via gastrointestinal absorption, as this avoids the possibility of their degradation in the intestine in inactive components;

- in addition, the concentration of isoflavones may be higher in topical applications when for certain effects (antioxidants, cell differentiation) oral administration is doubtful;

- also the treatment can be localized for topical applications in different dermatological diseases such as psoriasis, eczema, and the presence in the mixture, isoflavone alloys and other natural components such as phospholipids, polyphenols, sitosterols and saponins is a benefit for the skin.

Due to the absence of unwanted side effects, isoflavones could be used in topical applications to prevent skin aging, cellulite occurrence.

Zerykier, in the patent DE10329004 13.01./2005, highlighted the positive effect on the increase of the collagen quantity of the isoflavones, due to the similarity of the steric structure - with their two phenolic rings - with the structure of the steroid ring of estrogens (estradiol, estrone, estriol). Thus, having a substantially lower affinity than estrogen, genistein, daidzeine and formononetin have a slight estrogenic action, without causing their systemic or negative side effects. Genistein offers protection against UV radiation that causes wrinkles, and daidzein has a cytokine effect, energizing the cell growth that is protected and controlled by genistein.

- Patent application WO 200503015.7 (Al), proposes a series of cosmetic products based on isoflavones and bichochalcones for removing sebum from the skin, treating comedoomas, acne prophylaxis and seborrhea control. The isoflavones used can be: genistein and daidzein and clover, prunetine, biochanin A from clover and naut and pratensein from clover. The concentration used is between 0.05-0.5%.

- Patent application

US 2006002885 (Al) proposes a composition for the protection of sensitive, aged skin, for the natural growth of the skin barrier against the influences of the external environment, whose active ingredients are one or more bioquinones and one or more. ς * / 'treatment and prophylaxis of cellulite, firmness and reconstitution of function ^ -2012-00318-2 3 -08- 2012

isoflavones from the group: daidzein, genistein, genL-tina pruncnna) iochar: na A, -robei, santal. pratenseina; preferred is genistein, using a soybean Glycine preparation containing 0.05-2% and soy saponins with a concentration of 5-20%. In the finished product, the concentration of saponins is between 0.001-2%, of isoflavones 0.001-10%; the isoflavone: bioquinone ratio is between 50: 1-1: 50. Formononetin is not among the isoflavones used.

- Patent FR 2740681 / 09.05.1997 proposes a cosmetic composition with anti-cellulite action that contains: caffeine and an extract of the bark or leaves of the Vibumum plant; as active components, mention is made of flavones and apigenin units, but it is not shown by tests that biflavonoids are responsible for this effect.

- Patent FR 2848853 (Al) / 25.06.2004 claims a pharmaceutical composition, dietary for the treatment of post menopausal syndrome consisting of a mixture of soy isoflavones, soy saponins and vitamin K. Soybean titrated extract contains 26% saponins and 15% isoflavone.

- Patent application US 2005037099 (Al), proposes obtaining by hydrolysis and purification of a composition of soy, clover, kudzu and combinations thereof, for the treatment of wrinkled, depigmented skin, with acne, cellulite, sensitive, psoriasis, eczema. The soy composition can be enriched with sitosterols, saponins, phospholipids, coumestrols. The composition contains isoflavones genistein and daidzein. No information is given on the chemical concentration / characterization of the product obtained by hydrolysis and purification, nor the yields obtained.

- Patent WO 9918927 (Al) / 02/04/1999 claims a cosmetic composition using a clover extract, obtained by extraction with ethyl alcohol at 15:85, conditioned in butylene glycol. In the finished product, the extract can be in the concentration between 0.01% and 50%; no data are given regarding the chemical composition of the extract, the yields obtained. It is mentioned that the HPLC analysis revealed the presence of the molecules belonging to the isoflavones, such as fkgenistein, daidzein, biochanin, pratensein and coumestrol.

- Patent Application MX 201106224 (A) - Patents a composition containing isoflavones for treating symptoms related to menopause, obesity, cellulite, stress, insomnia, consisting of one or more isoflavones, a probiotic and a plant extract. Isoflavones can be found in the proportion 0.01-99.98%, and can be selected from: puerarin, daidzine, daidzein, genistine, genistein and their mixture, obtained from plants: Pueraria, Soybean, Trifolium.The composition is presented in liquid form or solid.No data on the composition of isoflavone preparations are given, biochanin A and formononetin are not mentioned.

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- Patent application US 2002106388 (Al) - proposes a cosmetic formula for the treatment of cellulite containing flavones and isoflavones. The treatment is directed towards the control of collagen and the reduction of the fat mass. The isoflavones used are genistein and daidzein, supplied by Merk, of min purity. .99.5%, added in a proportion of 0.1-0.2%, together with hydroxyphlavon-quercetin, a xanthine derivative-theophylline, camitin and a Coleus plant extract.

- Patent application KR 20020063479 (A) - claims a cosmetic and pharmaceutical composition containing isoflavone-genistein, in the dose of 1.5-120 mg / day as the active substance, with effect in the treatment of eczema and seborrheic dermatitis. In addition to isoflavones, the composition also contains proanthocyanidins, glucans, squalene and a tea extract.

- Patent WO 0164177 (Al) / 07.09.2001 proposes for the treatment of cellulite a mixture of free or glycosidated flavones and isoflavones obtained from soy proteins, folding from commercial preparations, or using various plants, such as red clover. , lentils, peas, by extraction with one of the solvents: ethanol, isopropanol, ethylene glycol, propylene glycol, butylene glycol, or in mixture with water. According to the invention, the pharmaceutical and cosmetic preparation contains a total of 0.1-20% of flavones, and their isoflavones or glycosides a percentage of 0.0001-3%, preferably 0.005-0.5%, in female products; the claimed isoflavones are not biochanin and a concentration of the isoflavone and flavone preparations used is not specified, but only their proportion in the finished product.

Proanthocyanins and proanthocyanidins are colorless flavonoid compounds that form with anthocyanidin mineral acids. The first proanthocyanidins were extracted from uncooked black grapes, white ripe grapes and young leaves of Vitis vinifera. Hydroxylated or hydroxylated products are produced as a chemical structure, and the dc is derived from the 3-4- diol yellow flask.

- The patent EP 2 165 712 A1 / 24.03.2010 proposes the use of a product characterized by a content of at least 50 mg / g phenolic compounds-procyanidins, propelargoidines, prodelfinidines in cosmetics as an intermediate or additive for antioxidant capacity, or in pharmaceutical compositions. for antiglycemic action in the treatment of diabetes, in urinary infections for antimicrobial capacity.

It has been established that proanthocyanins have free anti-radical action 20 times more active than vitamin E and 50 times higher than vitamin C. Proanthocyanins act synergistically with vitamin C by enhancing its absorption. Thus, proanthocyanidins can be used in cosmetics, for capillary protection effects [16], skin protection and as elastase inhibitors [17]. Also, proanthocyanidins have an incredible ability to maintain the collagen structure.

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which acts on the enzymes c: it causes the collagen, elastin and hyaluronic acid to decrease, so direct action. The second mechanism involves the neutralization of lipid peroxidation of cell membranes by free radicals.

The specific actions of proanthocyanidin A ₂ separated from wild chestnut seeds were highlighted by pharmacological tests. It has been found that proanthocyanidin A ₂ does not exhibit significant acute toxicity when administering po and ip. It also presents a significant healing action compared to the triterpenic fraction obtained from Centella asiatica, a well-known scar used in European countries. A vasoprotective and anti-ulcer activity has been observed, which seems to be a consequence of the protective activity in the microcirculation level. Tests performed both in vitro and in vivo showed anti-lipid peroxidant action inhibiting malondialdehyde (MDA) formation. Proanthocyanidin A ₂ can be used in various pharmaceutical formulas: capsules, tablets, dragees, solutions or suspensions for oral administration, injectable forms, suppositories, ointments, emulsions. [18] A preliminary study [19] performed with proanthocyanidins extracted from grape seeds, at a dose of 150 mg / day, determined the increase of capillary resistance in subjects with hypertension and diabetes. In the double blind experiment with a combination of two flavonoids: diosmin and hesperidin, in six weeks the symptoms of capillary fragility were reduced. The use of vitamin C together with flavonoids is recommended for capillary fragility.

Triterpene saponins

From a phytochemical point of view, among the bioactive compounds of plant origin the most studied are the class of pentacyclic oxygenated triterpenoids with chemical structure type ursan and oleanan, in free or glycosidated state, in the form of a complex of triterpenic saponins, named after the name of the triterpenic aglicon or of the plant from which they come: - oleanozides aglicon oleanolic acid from Calendula off aralozide - aglicon oleanolic acid from Aralia species; ginsenozide - aglicon oleanolic acid + protopanaxadiol + protopanaxatriol from Pcmax ginseng .; hederasaponine-aglycone oleanolic acid and hederagenin from Hedera sp .; triterpenic saponins-scina in Hippocastan semen;

There are numerous studies that highlight the biological effects of saponins on higher organisms. Thus, saponins increase the permeability of cell membranes, forming pores [20], The haemolytic action of saponins appears to be the result of aglycone affinity for membrane sterols, mainly cholesterol, with which they form insoluble complexes. The amount of glycosides required for permeabilization is much lower for cholesterol-rich lipid layers than for those without membrane cholesterol. [21] Oysters show that the interaction between saponins and membranes is much more complex,

CV- 2 o 1 2 - o 3 3 1 8 - 2 3 -08- 2012 the interaction being independent of the presence of cholesterol. In general, saponinelc have been studied for the benefits related to lowering cholesterol, stimulating immunity and inhibiting cancer cells. blocking cholesterol in the intestine, both food and liver, and eliminating it without being resorbed.

Indena cosmetics company conducted an activity and efficacy study with saponins isolated from wild chestnut seeds. Being very good surfactants, chestnut saponins are recommended as delicate cosmetic agents for foaming, which can be used to reduce aggressive and irritating chemical agents for the skin.

Efficacy experiments for the treatment of venous insufficiency with different saponins and sapogenins separated from Hippocastani semen and Hedera helix were performed in vitro by inhibition of specific enzymes. [22] Thus, only sapogenins from Hedera helix - hederagenin and oleanolic acid - showed comparable values of non-competitive inhibition of hyaluronidase, concentration dependent. They had the same behavior towards the elastase. Constituents from Aesculus hippocastani inhibited only hyaluronidase, the most important effect being saponin 1-spine. Genin escinol was less active.

The triterpenic saponins complex existing in the plant Hedera helix (ivy) family Araliaceae has predominantly hederagenin aglycone, along with oleanolic acid.

Hydroalcoholic extracts from Hedera helix are the basic component of many antitussive pharmaceutical products provided in the European drug nomenclature, [23] and glycolic extracts are used to obtain anti-cellulite and antilipemic dermatocosmetic products.

- The patent GB 1106133 / 13.03.1968 shows the obtaining of extracts from Hedera helix: saponin, sapogenin and magnesium salt of sapogenin which are inhibitors of cell division and growth having no mitoclastic effect.

- Patent GB 2051575 / 08.01.1980 discloses obtaining pharmaceutical compositions based on ivy extracts (Hedera helix) containing: Hederasaponin C (Hederacozide C) 60 ..90% or α-hederin, with the possibility of using tablets, solution for injection, ointment, as drugs with antifungal and antiparasitic activity, in human and veterinary therapeutics.

- US Patent 20060057236 / 16.03.2006 / Al shows a process for obtaining a leaf extract of Hedera helix, which contains α-hederine in proportion of 4.7%, and can be conditioned in the form of pharmaceuticals for use internally and externally, applicable in the treatment of respiratory disorders, due to the bronchospasmolytic effects of α-hederin. ~

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- Patent CN 1245693 / 01.03.2000 discloses c medicamc powder for the treatment of various forms of leukemia, which contains as main material the ivy powder, together with other pulverized plant products, the therapeutic effect having a cure rate of 96%, without toxic side effects.

- Patent RU 2127605 / 20.03.1999 presents a method of stimulating cellular immunity using triterpenic glycosides isolated from different plant organs of the Hedera genus as an adjuvant bioactive product, the method being applicable in the preparation of vaccines.

polyacetylenes

Brusts are traditionally used for topical use for eczema, psoriasis, alopecia, acne, boils, abscesses, local skin infections but not enough scientific data has yet been gathered to prove these effects through the mechanisms of action.

The roots of the bracts contain about 27-45% inulin, mucilages, 0.06-0.18 essential oils, sulfur and aliphatic antibacterial polyacetylene, bitter substances, polyphenols: caffeic acid, chlorogenic; volatile acids. Therapeutic efficacy in phytotherapeutic practice has so far been attributed to mucilages and inulin.

The desmutagenic activity of the breast was evidenced by in vitro studies against various mutagenic agents - 4-NO2-1, 2-DAB, ethidium bromide and was attributed to arctigenin. [24]. There are also studies that suggest that freshly squeezed juice inhibits the development of abnormal chromosomal changes induced by DMBA. [25, 26], the subcutaneous administration of a crude root extract of the root, in the experimental model determined the decrease of the induced edema, as well as the free anti-radical activity. [27]. Preliminary in vivo studies evaluating the activity of polyacetylene such as falcarinol, dehydrophalcarinol and dehydrophalcaridinol in mouse melanoma have demonstrated the antitumor potential of these compounds, the most active being the most effective.

There are studies that have shown antibacterial activity of broth roots that has been attributed to the presence of polyacetylene against gram-negative bacteria including E. coli, Shigella flexneri, and Shigella sonnei [28], aliphatic acetylenes isolated from broth roots are biosynthesized by the broth roots. - acetylene by two β-oxidations linked to C14 acetylenic precursors.

Acetylene forms a distinct group of natural products with relative chemical reactivity, which have been found in about 24 families of higher plants. Most acetylene in food plants are of the aliphatic type - falcarinol and belong to the family Apiaceae and Araliaceae. / And [

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The antifungal action of falcarinol st fidcarindiol has been shown on Botrytis spores cinerea and Mycocentrospora acerina that accumulate in carrot roots when they are stored. [Harding; Hale 1980], Plants of the Solanaceae family do not produce normally acetylene. and healthy, but when infected with molds of the Cladosporium fulvum type they accumulate acetylenic phytoalexins of the falcarinol type. [De Wit; Kodde 1981J. The antimicrobial properties of these compounds have also been explored against bacteria of the type Staphylococcus aureus and Bacillus subtilis.

Also, falcarinol and falcarindiol also exhibit anti-inflammatory and anti-platelet aggregating action. [Teng, 1999; Appendix 1993; Alanko 1994], For falcarinol it has been suggested that pharmacological action is related to the ability of the compound to modulate catabolism of prostaglandins by inhibiting the enzyme 15-OH-prostaglandin dehydrogenase [Fujimoto 1998],

- Patent EA 001339 (Bl) / 26.02.2001 presents an antimicrobial treatment for the treatment of infection with Herpes Simplex virus, in the composition obtained from the Echinaceae plant entering several phytochemical compounds: echinacosides, caffeic acid esters, enzymes, glucuronic acid, inulin , polyacetylene;

- Patent HU 212538 (B) / 29.07.1996 presents an active ingredient containing polyacetylene from plants of the family Compositae, Umbeliferae and Araliaceae, having acaricidal and aphidic action;

- Patent JP 3200736 (A) / 02.09.1991 proposes a new compound with polyacetylene structure, used as an antitumor drug, obtained from Ginseng species.

- Patent JP 3017043 (A) / 25.01.1991 claims the use of a new polyacetylene compound obtained from the plant Bluperum falcatum, as an anti-allergic, anti-inflammatory drug for oral use inhibiting 5-lipoxygenase or cyclooxygenase.

The following are the patents of the invention and the patent applications which present processes for obtaining bioactive products applicable in therapeutics, based on triterpenic glycosidated compounds, isoflavones, flavones, obtained from the plants Hedera helix, Hippocastani semen, Trifolium pratense lappa, and Centaureae cyani.

From the analysis of the literature data regarding the processes for obtaining extracts of Hedera helix, the following patents have been identified:

- Patent FR 1.531.621 / 24.05.1967 "Process for the preparation of triterpene complexes in plants" shows the obtaining of four strains of Hedera helix from saponin complexes in the form of oily liquids and a yellowish brown powder which have been characterized by thin layer chromatography showing the presence of 2-10 spots of saponins jn t

Ο 1 2 - Ο Ο 6 1 8 - 2 3 -08- 2012 each test. The process of obtaining provides for the extraction of the vegetable raw material with 60% ethanol and the processing of the total extract obtained in the form of powder by successive extraction with 96% ethanol, acetone, petroleum ether, methanol, and the purification step is carried out by adsorption on activated carbon in the ratio. 1/4 extract / adsorbent and subsequent desorption with different solvents: methanol, chloroform, ethyl acetate, benzene, pyridine. The major disadvantage of the patent is that the products obtained which are complexes of saponins are not analytically characterized by their content in the active substance, but only by methods of spot detection by thin layer chromatography. The process is laborious with many extraction and separation stages, it is uneconomical, obtaining small extraction yields even when mixing triterpenic compounds (from 100 kg plant between 4-450 g oily liquid extracts), using various solvents. slightly flammable, carcinogenic and non-ecological extraction and purification (benzene, pyridine, methanol, chloroform).

- The patent GB 1 106133 / 13.03.1968 shows the obtaining of extracts from Hedera helix: saponin, sapogenin and magnesium salt of sapogenin, by extraction with 70% ethyl alcohol, precipitation of the saponin complex with ammonium sulphate, purification with ethyl alcohol 93% and 98% methanol, and for the removal of pigments, resins and oils, acetone, ethyl acetate, methyl ethyl ketone, ethylene trichloride, chloroform are used by dissolving the concentrated sapogenin extracts as a dry residue followed by filtration for solvent removal. organic. In this way saponin is obtained, in the form of an amorphous powder, of a white-cream color, slightly soluble in water, methanol, ethanol, but insoluble in acetone and ethyl acetate, having a melting point between 195-200 ° C., And sapogenin as a white powder, insoluble in water at acidic pH, soluble in methanol, ethanol isopropanol.

- The patent application CN 200710034341 / 29.01.2007 shows the obtaining of a preparation of 98.2% Hederacozide C of purity determined by HPLC, by the hydro-alcoholic extraction 30-95% at reflux, obtaining the crude extract, dissolving it in water in a ultrasonic generator and subsequent extraction with ether and ethyl acetate to remove ballast substances, which impurities the extract. The extract thus obtained is extracted with n-butanol, and the purification is continued with 95% ethanol, ethyl acetate (to dissolve the insoluble part) methanol, and column chromatography using chloroforformethanol 65:35 as the eluent. The process is uneconomical, it cannot be applied on an industrial scale using expensive installations (ultrasonic generator, chromatographic columns), solvent mixtures difficult to recover and used and the obtained product is obtained with extremely low yields for applications in the pharmaceutical industry 0.21 g / 100 g vegetable raw material.

A

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- GB Patent 2051575 / 08.01.1980 entitled. "Pharmaceutical compositions containing sticky ivy extracts" means obtaining pharmaceutical compositions based on ivy extracts (Hedera helix) containing: Hederasaponin C (Hederacozide C) 60 ... 90% or α-hederin. The process for obtaining the extracts provides successive extraction of the plant with acetone and methanol, followed by precipitation of hederasaponne C from the methanol solution by treatment with ethyl ether. The result is hederasaponin C, with a content of 60%, which is purified by column chromatography of AI2O3 eluted with methanol, from which is obtained purified hederasaponin C, with a content of 90%. The conversion of hederasaponine C - 90% into ahederine is also presented. , by alkalizing with NaOH or 2N KOH, resulting in purified α-hederine.

- US Patent Application 20060057236 / 16.03.2006 / Al "Process for the production of an extract of ivy leaves", presents a process for obtaining a leaf extract of Hedera helix, which contains α-hederin, which provides for fermentation of the leaves. of ivy, with water at 30 ° C, when hederacozide C is enzymatically transformed into α-hederin which is extracted from the plant with hydroalcoholic mixtures (ethanol) to obtain a sicc extract, in powder form. It is mentioned that by total enzymatic transformation of hederacoside C from the plant in α-hederine, the sicc extract obtained according to the invention is enriched in α-hederine from 0.53% to 4.74%.

- Patent application US 20060210660 / 21.09.2006 / Entitled: "Process for the preparation of an extract of ivy leaves", presents a process for the preparation of an extract of ivy leaves, consisting of a mixture of 2 processed extracts separately for enrichment in hederacozide C and α-hederin. The extract enriched in α-hederine is obtained by the enzymatic fermentation of the ivy leaves with water, at 30 ° C, in order to convert hederacozide C into α-hederine which is extracted with ethyl alcohol 30% hydroalcoholic extracts being processed as sicc extract in the form of powder, with a rich content in α-hederin: at least 5%, due to the total conversion of the C hederacozide existing in the plant. The extract enriched in hederacozide C is obtained by treating the plant with superheated water vapor at 120 ° C, in order to inactivate the enzymes that convert hederacozide C to α-hederin, after which extraction is carried out with ethyl alcohol 30%, the hydroalcoholic extracts being processed as extract sicc with high content of hederacozide C: minimum 10%. In order to be used in therapeutics, a mixture of the 2 extracts mentioned above is prepared, obtaining a special final extract with a content of 5 ... 8% hederacozide C and 3 ... 5% a-hederine. Most patents of the invention relating to Hedera Helix show the use of glycolic, hydroglycolic, and hydroalcoholic extracts in the preparation of pharmaceuticals and

A

A- 2 Ο 1 2 - OO a 1 8 - 2 3 -08- 2012 cosmetics in the form of gel, cream, conditioner but also anti-amatory, antipruritic, antiparasitic, antilipemic and anti-cellulite, bronchospastic and to stimulate cellular immunity but which have some disadvantages:

- the processes are laborious, having many extraction and separation stages, they use different flammable extraction and purification solvents, carcinogens (benzene, pyridine, methanol, chloroform), they are uneconomical obtaining low extraction yields for applications in the pharmaceutical industry 0, 21 g / 100 g vegetable raw material for purified products and for mixtures of triterpenic compounds from 100 kg plant between 400-450 g oily liquid extracts).

- the processes cannot be applied at the industrial scale using expensive installations (ultrasonic generator, chromatographic columns, expensive adsorbents AI2O3), solvent mixtures difficult to recover and use;

- uses highly flammable and explosive solvents - ethyl ether - to obtain C 60% hederacozide by precipitation from the methanolic solution, resulting in a precipitation medium consisting of ethyl ethanol-methanol, difficult to distill and separated at industrial level in order to recover and reuse the solvents;

Hippocastan semen

From the analysis of the literature data regarding the procedures for obtaining the extracts of Hippocastani semen were identified the following:

The dry extract from Hippocastani semen, obtained according to DAB 10 and PE, comes in the form of a yellow brown powder, standardized in the 20% stage but which is not accepted for oral use, due to the traditional use it is recommended for external use for chronic varicose insufficiency, syndrome which includes swollen and tired legs, varicose veins and varicose ulcers. In the EMEA / HMPC / 225319/2009 Monograph there is the dried extract obtained from fresh chestnut seeds (40-80% v / v ethanol) standardized so that it contains 16-28% glycosidated triterpenes calculated as a herring. For skin administration it is suggested that the extract correspond to 100-150 mg stool / day, and the gel / ointment base should be 0.7-2% stool; There is a process of isolation at the laboratory level [29] of two proanthocyanidins: proanthocyanidin A2 and B2 (dimers) by extraction of fresh chestnut bark with methanol in a Waring reactor. The methanolic extract from which the lipid fraction was removed by extraction with petroleum ether is concentrated under reduced pressure and temperature below 30 ° C. The resulting concentrate is taken up with water and extracted with ethyl acetate and concentrated under low pressure. After dissolution in the methanol it is subjected to chromatography on the polyamide resin column (Polyamide Woelm) using as methanol eluent. Collect fractions which contain proanthocyanidin, concentrate and evaporate to dryness to obtain a crude proanthocyanidic preparation. It is dissolved in ethanol and subjected to chromatography on the Sephadex LH-20 column using ethanol development agent. Two proanthocyanidin fractions are obtained which are concentrated and evaporated to dryness resulting in proanthocyanidin B ₂ and A ₂ in the form of yellowish-white powders. These isolated and purified proanthocyanidins can be used either as they are or after dissolving in water, alcohol or alcoholic solution being added as antioxidants in different products: food, pharmaceuticals, cosmetics, lubricating oils, plastics, etc.

US Patent 3,766,166 / 16.10.1973 proposes a process for obtaining water soluble stage, from any type of dry chestnut extract, by adsorption chromatography; The dry extract was dissolved in a mixture of n-propanol: ethyl acetate: water in well-defined ratios, followed by passing the solution over activated aluminum oxide; the eluate containing the soluble spine is dried and washed with acetone; the purified stage obtained is presented as a white, amorphous powder, which is characterized only by mp = 218 ° C; the yield obtained according to the type of dry extract used is between 13,5-32,2%;

The patent RO 71026 / 16.03.1981 proposes a process for obtaining the crystalline stage of pharmaceutical purity by extracting the seeds of Aesculus Hippocastanum with a 65% water-miscible organic solvent - methanol, ethanol. The obtained extraction solution is passed on a column with cationic ion exchangers; the eluate leaving the column should have a pH of 3.5-3.8, concentrate in vacuo, and mix with water at which time the stage acid precipitates as a crystalline substance which is separated by filtration / centrifugation; for the passage in the form of salt-escinate, it is treated with a solution of sodium bicarbonate or triethanolamine; the sine or escinate of sodium have a purity of min. 97%, with max. 3% free stinic acid, being obtained with a yield of 1,5-2% compared to the substance introduced in the process.

US patent application 2006 / 0030697- shows the obtaining of the β-chain from Aesculus indica, by extraction with an aqueous alcoholic solution - methanol, ethanol, in a ratio of 1: 1-8: 2, the partition between the aqueous phase and an immiscible alcohol of preferred n-butanol or n-propanol, treating the organic phase with a 0.5-1% NaOH solution, separating the organic phase, passing it on acidic alumina; the organic eluates containing β-escina are concentrated until an amorphous powder is obtained, of purity 90-95%., with a yield of 2-3%.

GB patent 1,116,890 / 12.06.1968 claims a process for the preparation of chestnut-soluble saponins, by converting β-scene into water-soluble form α-stage;

the process can be done both starting from fresh plant material-seeds of Aesculus ¹ sA lUU-iA

Ο Ι 2 - 0 Ο 6 1 Β ~ 2 3 -08- 2012

Hippocastanum, as well as from β-escina, by 60% hydroalcohol extraction with methanol or ethanol, at room temperature, the extract obtained is concentrated and a NaOH precipitating agent is added, the pH reaches 6.7, the solution is passed through a column with cation-exchange resins, the obtained acid effluent is heated to 90 ° C for a short time, the soluble stage solution is centrifuged to remove the unconverted β-stage precipitate, concentrated and dried, gravitationally determining the amount obtained- 85 % water-soluble saponin in the form of a-stool;

GB 933,657 discloses a process for obtaining 2 isomeric a and β isomers from the natural one, which consisted of converting the one to the free acid form, maintaining the one-way solution in this form at a temperature between 50-90 ° C, up to when the βescin precipitate is completely formed and the solution of α-escina is separated by filtration / centrifugation. The liquid is concentrated and dried, 2 white powders a and β escina are obtained, characterized by specific m.p., by refractive indices and by the different degree of solubilization.

GB 929816 claims the isolation of the stage from a 10% chestnut hydroalcoholic extract using for precipitation an ethereal cholesterol solution, maintained at 90 ° C, the precipitate saponin-cholesterol is washed with water, dried it is extracted with ether, then with methanol, the alcoholic solution obtained is purified with activated charcoal, and by concentration under vacuum the sodium escinate is obtained 8%.

GB 1 539 127 / 24.01.1979 claims a process for obtaining the chestnut fruit stage by extraction with a mixture of aliphatic alcohol saturated with water and water saturated with aliphatic alcohol usually n-butanol, at pH = 3-4 , the separation of the organic phase and the extraction of the stage from it with an aqueous alkaline solution, so that the pH is between 5-5,5. The extraction of the stage in the aqueous phase is facilitated by the addition of ethyl acetate. The solution of sodium or potassium escinate is formed, which, after discoloration, evaporates and is concentrated under vacuum. The water-soluble ladder in acid form is obtained by dissolving it in methanol and passing it on a column with cation-exchange resins; a white powder with Rf identical to that of the standard is obtained, mp = 220-225 ° C, and the specific rotation angle. Another criterion of quality of the obtained stage is the 48-hour stability of the 10% solution in water.

CA 921404 / 29.02.1973 claims to obtain a rich concentrate in the stage, with a content of 40-50%, used as an active ingredient for obtaining capsules, tablets, dragees, ointments, suppositories. Aqueous acid extraction is used, followed by purification with a mixture of ketone alcohol, water

CA 674987 patent claimed the obtaining of the two isomers of the stage, by / ¹ * forming an adduct with cholesterol, separating the precipitate formed, transforming it into the stage.

iV / u / 4 ζγ- 2 0 ι 2 - 0 0 a 1 8 - -. _?

3 -08- 2012 of sodium, separation of isomers on cation exchange resins and centrifugation; 2m distinct fractions of the 2 isomers are obtained.

EP patent 1314432 / 28.05.2003 claimed to obtain a mixture of stairs 95

99.5% β and 0.5-5% a through which the milled chestnuts are extracted with methanolic solution at 40 ° C, with strong agitation, acidified with oxalic acid and the precipitate formed is dissolved in ethyl alcohol at 55 ° C. The supernatant is stirred, passed through a column with cation exchangers, the eluate is concentrated, acetone is added, maintaining for 30 minutes at 55 ° C and overnight in the refrigerator. A cyst concentrate is obtained, in which β-cyst represents 97% and 0.4% ash.

EP 0298 148 / 11.01.1989 claims to obtain an enriched β-escina preparation, starting from fruits / chestnuts preserved by freezing, they are kept for 30 minutes in an aqueous solution containing preservatives (peroxy-monosulfate). potassium, sodium benzoate, acetic acid) after which the chestnuts are sliced and extracted with water. The aqueous tincture thus obtained is centrifuged, acidified with sulfuric acid pH = 2.5-2.8, when the precipice precipitates; for purification, the chain is dissolved in methanol and discolored with activated carbon. The β-stain of purity 88.1% is obtained with a yield of 1.78% relative to the quantity of the raw material.

Compared to the patent proposed by us, the processes for obtaining extracts from Hippocastan semen, according to the above patents, have some disadvantages and differences: - they use very flammable solvents: ethyl ether or toxic: methanol;

- use for separation of columns with ion exchangers, or acid / alkaline media, or use cholesterol for adduct formation;

- aim to obtain purified or concentrated preparations in saponins-escina, considering flavones or proanthocyanins as impurities;

- often the obtained preparations are characterized only by qualitative data: Rf chromatographic, solubilities in different solvents, not quantitative.

From the analysis of the literature data related to the processes for obtaining the extracts of Trrifolium pratense, the following patents have been identified:

- Patent CN 1683381 (A) / l.9.10.2005 presents a simple process for obtaining an extract obtained from red clover grass, by extraction with alcohol and purification; HPLC analysis determines a continuum of not less than 12% formononetin and soforicol;

- GB patent 2483934 (A) / 28.03.2012 shows the obtaining of a botanical extract using an aqueous system of subcritical extraction, at a temperature between 150-200 degrees Celsius;

- Patent WO 99/48496 discloses a process for obtaining a composition containing 9% biochanin, formononetine 8%, genistein 1.5% and daidzeine 1%, by extraction with water at 25 degrees Celsius, for 3-4 hours, under nitrogen atmosphere, addition of ethyl alcohol, boiling 3 hours; extraction with heptane, finally obtained from 500 g plant 22 g. extract. The process is not economical, it is not ecological and it cannot be applied industrially, using atmosphere of nitrogen and slightly flammable solvents.

- US Patent 7,033,621 / 25-04-2006 claimed to obtain a composition containing 36-70% isoflavones: genistein, daidzeine, formononetine, biochanin and glycytine by water extraction, in which hydrolysis and aglicone supplementation are supplemented. with enzymes; extraction of aglycones is performed with ethyl acetate, maintaining a sufficiently long time so that the free isoflavones pass in the organic phase.

- Patent CN 101239961 / 13.08.2008 proposes a method of obtaining isoflavones through a catalytic and hydrolysis operation using a strong acidic ionic liquid, with good thermal stability and a solid layer of aluminum oxide or silica gel; the hydrolysis reaction is performed in a proportion of 100%;

- Patent CN 101407507 / 15.04.2009 presents a method of separating isoflavones from primary extracts that involves the use of macroporous resins AB-8. The primary extract is dissolved in methanol and placed in a chromatographic column. The isoflavones in the primary extract are absorbed by the macroporous resins, some of the impurities are removed by elution with an alcoholic solution of 10-20%, after which the isoflavones are eluted with an ethanolic solution of 65-75%. The combined eluates were concentrated in vacuo to give a yield of 0.25-0.35 g isoflavone / 1 g primary extract.

- Patent CN 101766673 / 07.07. 2010 presents a general method of extraction of isoflavones from an oriental plant consisting of the primary extraction in the form of aqueous decoction, followed by alcoholic extraction, concentration for the removal of organic solvent, removal of impurities by extraction with cyclohexane, dissolution of the residue by heating in alcohol, discoloration with hot coal, filtration, concentration, drying, grinding, obtaining an isoflavone rich extract which is conditioned by mixing with PEG 4000, 6000, polyacrylic resins, magnesium stearate to be encapsulated;

- The patent application US 2010048689 / 25.02.2010 proposes to obtain isoflavones from residues of the food industry - soy derivatives, using a fermentative enzymatic process with genetically modified Aspergillus strains, by which malonylated derivatives,

Ο 1 2 Ο Ο 5 1 8 - 2 3 -08- 2012

- Patent CN 101559094 / 21.10.2009 claims a process for extraction and separation of isoflavones from the red clover through a dynamic operation with high-grade alcohol solvent, filtration, concentration, followed by extraction with a solvent mixture, a moderate hydrophobic one. , polar and second weak polar, when the transfer of the active ingredients in the solvent mixture takes place, the concentration of phases, the addition of a weak polar solvent for crystallization, the precipitate is separated and processed by vacuum drying.

- Patent CN 1013866133 / 18.03.2009 presents a process for the extraction of isoflavones from the red clover with ethanol, followed by alkaline hydrolysis, neutralization, purification, extraction with ethyl acetate, the phase from which the isoflavones are separated by vacuum concentration.

- Patent CN 1709886 / 21.12.2005 claimed a method of extraction of the isoflavonoid total from the red clover by: extraction of the dried and minced plant with hot ethanol, in counter current, the extracts obtained are dried and the solvent is recycled; follows the second extraction with petroleum ether or other solvent to remove the oily lipid phase, drying the filtrate, adding ethanol, reextraction, purifying on neutral aluminum oxide, washing the filtrate, extracting with saturated ethyl acetate, concentrating the organic phase from which it is obtain the total isoflavonic.

- Patent EP 1391208 / 25.02.2004 proposes a process for the preparation of a plant extract with a continuous high in isoflavones of at least 40% which consisted of the use of a succession of solvents, thus in a first extraction with water, followed by the extraction with methanol, acetone or an aliphatic alcohol, ethyl acetate obtaining increasingly concentrated preparations in isoflavones. Use red clover leaves or fruits; all extraction methods can be used. The extract obtained can be dried by lyophilization; no information is given on the yield yield nor quantitative data of the isoflavonic components. It is noted that the total isoflavonoid is composed of daidzein, genistein, formononetin, biochanin A and glycosides ononine and sissostrine.

The procedures for obtaining red clover extracts, according to the above patents, have some disadvantages:

- are not environmentally friendly, using many toxic or flammable solvents: petroleum ether, heptane, cyclohexane, acetone;

- it is made in the heat of -150-200 degrees Celsius, so high energy consumption, or in expensive installations - supercritical system, in counter current;

- for hydrolysis, strong alkaline / acid environments or expensive enzymatic systems are used;

Ar 1 o 1 2 - 0 O a 1 a - 2 3 -08- 2012

- macroporous resins, aluminum oxide, chromatographic columns are used for purification - systems that are not suitable for lifting on an industrial scale;

- In general, no data are given regarding the yield or chemical characterization of the preparations, but only the total isoflavones are expressed or the components present are listed; Regarding the production of polyacetylene from vegetable raw materials, especially from Radix Bardanae, it is mentioned:

- The patent JP 3200736 / 02.09.1991 proposes a process for obtaining a polyacetylene compound of Ginseng species, by extraction with hexane, diethyl ether, petroleum ether, ethyl acetate, methanol. The solvent is removed and the residue is purified on a chromatographic column using polymers of the type Diaion (R) HP-20, Sephadex, Silicagel, polyamide.

- The patent JP 3017043 / 25.011991 proposes to obtain from Buplerum falcatum a polyacetylene compound, by extraction with hexane, distillation and recovery of the solvent, the obtained residue is chromatographed on a cellulose column, eluted with hexane, and by distillation eluate produced. used as a medicine.

- The patent JP 2172926 / 04.07 for obtaining a polyacetylene compound is made a supercritical extraction with carbon dioxide, to protect the triple bond, at 35-36 degrees Celsius and 100-300kg / cm ² , to improve the extraction yield. If necessary, further extraction with chloroform is carried out.

The following disadvantages result from the analysis of the above patents:

- no patents are known of the invention for obtaining polyacetylene products from Radix Bardanae;

- many flammable and toxic solvents are used, during the purification phases of the extracts, or expensive installations (supercritical);

From the analysis of the state of the art regarding the procedures for obtaining extracts from Centaureae cyanum herba, the following results:

- Patent LT 5784 / 25.10.2011 presents a cosmetic composition used for antioxidant and hydration effects achieved by combining at least 3 aqueous extracts between plants: Camelia sinensis, Ginko biloba, Glycyrrhiza, Centaurea cyanus, Ruscus aculeatus.

- The patent application KR 20100031068 / 19.03.2010 provides a cosmetic composition of hydration and softness obtained by mixing a series of plants in an optimal ratio; thus 0.05-1 parts of each plant - Borago off., Centaurea cyanus, lavandula off., Salvia off. stir and subject to hot extraction 80-120 degrees Celsius, followed by cooling the room temperature.

t

c \ -2 O 1 2 - O O 3 1 8 - 2 3 -08- 2012

- Patent JP 2007176877 / 12.07.2007 presents a medicinal composition for external use obtained by using at least one extract of: Bellis perennis, Arthemisia dranunculus, Centaurea cyanus used to prevent or ameliorate various symptoms that accompany inflammation, photo-aging of the skin.

- Patent JP 2005306768 (A) - Claims a method of inhibiting the production of melanin by using an external preparation, characterized by a mixture of extracts between: Sophora flavescens, Centaurae cyani, Achillea millefolium, Tanacetum parthenium.

- Patent EP 1632223 / 08.03.2006 proposes a complex of cosmetically active substances for skin regeneration consisting of a 5-20% Centaurea cyani extract and an 80-95% soybean extract which are included in a composition. cosmetics in the report

0.1-20%.

The use of blue extracts is quite low, the extracts are made / used in other ways / combinations than according to our process.

The patent solves the technical problem:

- to obtain some phytocomposites standardized in several active components, with complementary biological actions, in order to make their therapeutic applications more efficient. Thus, the extraction processes follow a well-delimited continuum of the following compounds associated in the final plant extracts: flavonoid compounds and isoflavonic aglycones; proanthocyanins and triterpenic saponins; triterpenic and flavonoid saponins; carboxylic polyphenol acids and triterpenic saponins; carboxylic and polyacetylene polyphenol acids; carboxylic polyphenol acids and flavonoid compounds.

- of ecological valorisation of the resources of medicinal plants, which implies the cultivation of plants in ecological system for obtaining the plant raw material necessary for the production of phytotherapeutic preparations;

- the extraction and separation procedures of the active components / extracts are simple obtaining optimum and not maximum yields according to the principles of ecological technologies, with the use of all resources; by the technological parameters applied in the extraction plant a high speed of separation of the components of the plant is printed due to the placement inside the loop of high speed centrifugal pumps causing a reduction of the extraction time from 4 hours to 2-3 hours / stage;

- cosmetically active preparations / ingredients are standardized in the active principles responsible for the biological action followed and proven by specific tests, in vitro;

- obtaining selective extract called Dermo ET isolated from Herba Trifolium pratense, conditioned as a fluid extract, in glycerin, propylene glycol or butylene glycol

^ 2012'00318-2 3 -08- 2012 standardized in flavonoid compounds expressed in quercetin of min.0.15 g / 100 ml and in isoflavonic aglycones of min.0.23 g / 100 mL as the sum of daidzein, genistein, formononetin and biochanin A, quantitatively determined by HPLC, namely: daidzeine min. 10 mg / 100 mL, genistein min. 20 mg / 100 mL, formononetin min. 140 mg / 100 mL and biochanin A min. 60 mg / 100 mL, obtained in ratio 2: 1-1: 1 plant: final extract;

- obtaining the selective extract - isolated from Hippocastani semen, conditioned either in the form of fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo Cas standardized in proanthocanes of min.0,15 g / 100 g and total triterpenic saponins expressed in min stage .2 g / 100 g, obtained in 1: 1 ratio: final extract; or in the form of a dry extract in the form of a non-hygroscopic yellow-brown powder, called Dermo Es, standardized in triterpenic saponins expressed in min.70% scale, and in flavonoids of min.2% obtained in 33: 1 ratio plant: final extract ;

- obtaining the selective extract isolated from Hedera helix folium, conditioned either in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo EH, standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0,3 g / lOOg, and in Total triterpenic saponins expressed in hederacozide C of min.3 g / 100 g, obtained in 1: 1-1: 2 ratio plant: final extract, or in the form of dry extract in the form of a white-brown, non-hygroscopic powder, standardized in saponins triterpenes expressed in hederacozide C of at least 60%, obtained in ratio 20: 1 plant: final extract;

- obtaining the selective extract isolated from Radix Bardanae, with antimicrobial properties due to the presence of polyacetylene, conditioned in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo B, standardized in polyphenol carboxylic acids expressed in caffeic acid.0 min. / 100 g ,, obtained in ratio 1: 1 plant: final extract;

- obtaining selective extract isolated from Cyanus Herb Centaurea conditioned as a fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo Abs, standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0,9 g / 100 g and in flavonoid compounds routinely expressed as min.0,5 g / 100 g ,, obtained in ratio 1: 1-1: 2 plant: final extract;

- obtaining a cosmetic range through the original combination of phytotherapeutic preparations whose biological action proved by specific tests, cumulates the restoration effects of dermal, anti-edema and anti-inflammatory structures, namely:

- Anti-cellulite cream and gel

- Gel and cream with anti-dandruff effect

- Serum and anti-tar cream

¢ 2012-00318-2 3 -08- 2012

By applying this process, the therapeutic potential of the plant raw materials which is dried and subjected to a common primary extraction operation for all the raw materials made with ethyl alcohol of different concentrations depending on the intended component is technologically exploited at industrial level. between 40-96%; the obtained extracts are still subjected to processing by bleaching with activated carbon, vacuum concentration and conditioning in the appropriate cosmetic solvent, in the established ratio obtaining selective fluid extracts; In the case of dry extracts, after the concentration step under vacuum, the obtained product is subjected to selective separation with one of the appropriate solvents: nbutanol, ethyl acetate - the separate organic extracts are further processed by vacuum concentration, until the dry residue is purified. by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius; The standardized extracts thus obtained are subjected to chemical analyzes to determine the concentrations of active substances and the traces of solvents used are each conditioned by the purpose of their intended use, becoming pharmaceutical or cosmetically active intermediates.

The products obtained according to the present invention by successive extractions and separations of Trifolium pratense herba plants, Hedera helix folium, Aesculus hypocastanum semen, Radix bardanae, Centaureae cyanum herba have the following cosmetically active effects: anti-cellulite, anti-edema and anti-cear.

THE TESTS CARRIED OUT TO EVIDENCE OF THE SPECIFIC ACTIVITY AND EVALUATION OF THE APPLICATIONS IN THERAPEUTICS OF THE ACTIVE COSMETIC COMPONENTS OBTAINED FROM RED TRIFO, IEDERA, CASTAN AND BLUE

Demonstration of the biological effects of the plant extracts was performed at the target cell level, on standardized cell lines relevant to the studied mechanism:

• The anti-inflammatory effect at the vascular endothelium level was highlighted on the specific HUVEC line (human umbilical vein endothelial cells) due to its relevance in simulating the inflammatory process at the blood vessel level. The effect of dermal restructuring was tested on human dermal fibroblasts (HS 27). cell type responsible for the synthesis of structural proteins from the extracellular matrix, of which the most important are type I and III collagen.

• The photoprotective effect was demonstrated on the human keratinocyte HaCaT line, this type of cells forming the epidermis, the first layer of skin tissue, depending on the barrier against the harmful action of UV radiation.

^ -2012-00318-2 3 -08- 2012

Keratinocytes from cell line H; .CaT. after 24 hours. of accession, were treated with the substance of interest for 48h. Passages 57-63 were used, the standardized cell line being immortalized. The culture medium used was complete DMEM, with glucose supplement (1.35g / L), 1% antibiotic / antimycotic, 10% fetal bovine serum.

HS27 standardized human dermal fibroblast cell line was used between passages 15-22. Testing was performed on adhered cells for 48 h. In culture flasks of 12.5 cm ² , in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 1% antibiotic / antimycotic and incubated at 37 ° C in 5 atmosphere. % CO ?. The action of the substances was evaluated after 48h. incubation.

HUVEC endothelial line tests were performed between passages 2-9. The cells were grown in RPMI culture medium, supplemented with 1% antibiotic / antimycotic, 10% fetal bovine serum. After 24 h of accession, the cells were treated for 48 h. with vegetable extracts.

Demonstration of the dermal restructuring effect of Trifolium pratense extract

The effect was shown on cultures of human dermal fibroblasts (cell line selective for the effect pursued), by complementary data at the following mechanisms: stimulation of collagen synthesis and cell multiplication rate, as well as induction of overexpression of integrins αϊβΐ and α2β1 (restoring firmness -epidermice).

Clover extract, Dermo ET isolated from Grass Trifolium pratense, contains as active principles daidzeine, genistein, formononetin and biochanin A, so the activity of these components, as well as their combination in the corresponding doses of clover extract, was evaluated in its action demonstration. .

a) Stimulation of collagen synthesis

Collagen degradation in the extracellular matrix is largely due to the proteolytic activity of metalloproteinases (MMPs) that are expressed by both dermal fibroblasts and inflammatory cells, playing an important role in matrix remodeling of the skin undergoing a long-term inflammatory process. which has the consequence of losing the integrity of the matrix and diminishing the elasticity of the skin (30). Matrix metalloproteinases (MMPs) are poorly expressed in normal adult tissues, but are overexpressed by cytokines or growth factors in physiological and pathological processes. The regulation of the extracellular matrix implies a balance between the synthesis of its structural components and their degradation under the catalytic action of MMPs whose biological function is modulated by specific tissue inhibitors (TIMPs).

^ -2012-00618-2 3 -08- 2012

Substance Collagen pg OHPro / 2 * 10 cells / ml MMP MMP9 (pixels) MMP2 (pixels) Name Dose % of variation % of variation % of variation Cell witness 0.0712 90.84 94.26 Solvent control .0617 89.11 93.3 Dermo ET 1/1000 .1012 39.03 73.18 -21.8 94.61 1.4 1/2000 .0774 20:28 78.55 -13.4 94.32 1.1 daidzein 2.6 μΜ 0.0758 18.60 74.08 -20.3 90.61 -3.0 1.3 μΜ .0761 18.92 73.83 -20.7 89.69 -4.0 genistein 3.4 μΜ 0.0325 -89.85 76.21 -16.9 91.95 -1.5 1.7 μΜ .0287 -114.98 79.08 -12.7 92.98 -0.3 Biocianina 6.4 μΜ .0370 -66.76 81.52 -9.3 91.63 -1.8 3.2 μΜ 0.0436 -41.51 72.71 -22.6 89.96 -3.7 Formononetin 32.7 μΜ .0583 -5.83 71.96 -23.8 95.95 2.8 16.4 μΜ 0.0396 -55.81 77.11 -15.6 94.32 1.1 (Daidzein + Genistein + Biochani na + Formononetin) equivalent Dermo ET 1/1000 .0786 21.50 92.95 4.1 96.71 3.5 1/2000 .0455 -35.60 91.04 2.1 95.86 2.7

The experimental data presented in the Table above show that of the phytohormones studied only Daidzeina induces fibroblastic cells maintained under normal growth conditions, both type I and III collagen biosynthesis, as well as inactivation of the MMP9 proteolytic enzyme, thus helping to remodel the affected area. Even if the other phytohormones analyzed individually showed an inhibitory effect on matrix proteolytic enzymes, the process of collagen biosynthesis could not be stimulated in their presence, thus observing the decrease of the structural protein concentration in the growth medium. However, the introduction into the growth environment of phytohormones in different combinations potentiated by at least 13.5% the effects of Daidzein, an aspect observed also in the case of Dermo ET.

b) Stimulating the rate of cell multiplication

The effect of Dermo-ET on the cell proliferative status was tested by 2 complementary analysis techniques: cell cycle sequencing and sequence of proliferative generations (flow cytometry, propidium iodide labeling and CFSE - carboxy fluorescein diacetate succin imidil ester), respectively. The results were estimated as Proliferation Index, respectively the sum of the percentages

Ο 1 2 ~ Ο Ο 3 18-2 3 -08- 2012 cells in S and G2 / M multiplication phases. calculated with a specific analysis software (FACS

Express V3 module DNA cell cycle and proliferation).

Substance tested %phase S + G2 / M % of variation Proliferation index % variation WITNESS 18.5 2.4 Solvent control 19.5 2.6 Dermo ET 1/1000 28.9 48.2 3.5 34.6 Dermo ET 1/2000 25.3 29.9 3.0 16.9 Formononetin equivalent Dermo ET1 / 1000 48.4 148.2 3.2 23.8 Biochanin equivalent Dermo ET 1/1000 47.2 142.1 3.9 48.8 Genisteina equivalent Dermo ET 1/1000 17.4 -10.7 3.3 27.3 Daidzeina equivalent Dermo ET 1/1000 27.8 42.6 3.5 33.8 (Daidzein + Genistein + Biochanin + Formononetin) equivalent Dermo ET 1/1000 56.1 187.7 5.4 106.5 (Daidzein + Genistein + Biochanin + Formononetin) equivalent Dermo ET 1/2000 54.0 176.9 5.1 95.4

The results demonstrate the acceleration of the cellular multiplication rate induced by clover extract, Dermo ET, by over 30% compared to the corresponding control. The acceleration of the S phase and the entry into the mitosis is strongly influenced especially by formononetin and biochanin, daidzeine having a weak effect, and genistein is not active on this mechanism. In contrast, the ratio in which the 4 components are found is optimal for stimulating cell proliferation, which can be mutually reinforcing.

c) Induction of overexpression of α integrβΐ and α2β1 integrins (restoring firmness of dermo-epidermal structures)

Integrins are functional proteins consisting of 2 glycoprotein subunits (a and β) that extend along the membrane capable of binding multiple ligands, including molecules in the extracellular matrix, which play an important role in cell adhesion, movement and cell migration. Integrin αϊβΐ mediates the feed-back regulating collagen synthesis, making cell-collagen or cell-laminin-type connections in the extracellular matrix, and Integrin α2β1 mediates stimulation of type I collagenase (MMP1) with role in fibrillogenesis (collagen organization in fibrils), binds collagen to spikes. Balance between Integrin α Integrβίδΐ Integrin α2βIt is important to maintain the balance between collagen degradation and synthesis. [31, 32, 33].

The technique of integrins detection by flow cytometry involves using monoclonal antibodies for the α and β chains (CD49a, fluorescently labeled for PE, correspondingly

/

(^ -2012-00318-2 3 -08-2012 of subunit "2; CD49b, fluorescently labeled for FIT'C, corresponding to subunit of; and CD 29 fluorescently labeled for APC, corresponding to subunit βΐ).

The results (average of 3 experiments) are presented in the table below as the median of the fluorescence channels corresponding to the expression of the 3 glycoprotein chains:

Substance tested FITC-A Mean (CD 49b Integrin alpha2) % of variation IN-A Mean (CD 49a integrin alphal) % of variation APC-A Mean (CD 29 Betal integrin) % of variation WITNESS 14448 5837 4167 PG 15122 4.67 6330 8.45 4984 19.61 TGF beta 4ng / mJ 44217 206.04 7005 20.01 8123 94.94 Dermo ET 1/1000 29 921 107.09 4851 -16.89 3719 -10.75 Dermo ET 1/2000 25796 78.54 4092 -29.90 4314 3.53 Formononetin equivalent Dermo ET1 / 1000 14313 -0.93 3193 -45.30 2748 -34.05 Biochanin equivalent Dermo ET 1/1000 24491 69.51 3220 -44.83 3594 -13.75 Genisteina equivalent Dermo ET 1/1000 23269 61.05 3834 -34.32 4448 6.74 Daidzeina equivalent Dermo ET 1/1000 18954 31.19 3797 -34.95 3597 -13.68 (Daidzein + Genistein + Biochanin + Formononetin) equivalent Dermo ET 1/1000 27 371 89.44 6830 17.01 5308 27.38 (Daidzein + Genistein + Biochanin + Formononetin) equivalent Dermo ET 1/2000 13679 -5.32 6726 15.23 5489 31.73

The tests revealed the action of the selective extract of Herba Trifolium pratense, Dermo ET, in the dose-effect manner, only on the induction of the overexpression of the glycoprotein chain a2, indicating an amplification of fibroblast-type I collagen bonds and stimulation of collagenase activity with role in fibrillogenesis. It has been shown that this action is mainly given by biochanin and genistein in the extract component, and to a lesser extent by daidzein, formononetin not acting at this level. The determined effect is similar to that of the positive control (TGF beta 4ng / ml).

The results obtained by testing the clover extract through the three mechanisms of dermal tissue reconstruction demonstrate the complementary role of daidzein, genistein, formononetin and biochanin, as well as the importance of their combination in a certain ratio for maximizing the effect.

Λ- 2 Ο 1 2 - Ο 3 3 1 3 - 2 3 -08- 2012

Demonstration of anti-inflammatory activity at the vascular endothelial level of herbal preparations of Hedera helix, Aesculus hypocastanum, Arctium lappa and Centaur ea cyanus

Vascular inflammation is characterized by adhesion between lymphocyte and endothelium triggered by the expression of adhesion molecules - markers of inflammation and secretion of pro-inflammatory cytokines: ICAM, characteristic of microvasculature and VCAM, specific to large blood vessels [34]

The anti-inflammatory effect at the vascular level of the preparations Dermo -Abs, Dermo-B, Dermo-Es and Dermo-HdC was determined, compared to a positive control of Dexamethazone, a known anti-inflammatory agent, at a concentration of 0.6pg / ml. [35]

The "in vitro" simulation of inflammation was performed with LPS (polysaccharide of bacterial origin, to mimic the conditions associated with local infection) and TNFα (non-specific inflammatory stimulus).

The protein expression of VCAM-1 and ICAM-1 was evaluated by flow cytometry. (fluorescence labeling with antibodies suitable for ICAM-1 and VCAM-1, APC Mouse Anti-Human CD54 for ICAM-1 (intracellular-adhesion molecules) and PEMouse Anti-Human CD106 for VCAM-1 (vascular-cell-adhesion molecules) The results are analyzed by comparing the average of the APC fluorescence channel, respectively PE for the acquired samples, the results were compared with the secretion of human inflammatory cytokines (IL6 and IL8), expressed in pg / ml (marking and analysis with the BD Cytometric Bead Array kit (CBA). ) - Human Inflammatory Cytokines kit (BD Pharmingen) The results are presented in the tables below:

well tested unstimulated cells cells stimulated LPS lug / ml TNF alpha stimulated cells 2ng / ml ICAM % vary to you VCAM % vary ation ICAM % vary to you VCAM % varies to you ICAM % vary to you VCAM % vary to you Cell witness 10882.6 1366.9 13974.4 1287.0 15697.5 1569.7 Solvent control (PG) 10436.0 1448.0 11789.0 1499.0 11053.0 1552.0 Dermo-HDC ΙΟμΜ 12443.3 14.3 1315.0 -3.8 12626.3 -9.6 1316.3 2.3 13275.0 -15.4 1399.5 -10.8

f

^ -2012-00318-2 3 -00- 2012

Dermo-HDC 15μΜ 12101.1 11.2 1302.0 -4.8 12182.3 -12.8 1478.1 14.8 13241.2 -15.6 1199.3 -23.6 Dermo-HDC 20μΜ 11941.2 9.7 1285.7 -5.9 12414.6 -11.2 1463.4 13.7 12036.7 -23.3 1201.9 -23.4 Staircase 2μΜ 12494.9 14.8 1249.3 -8.6 13453.7 -3.7 1246.1 -3.2 16041.7 2.2 1491.2 -5.0 Staircase inaμΜ 12784.2 17.5 1348.1 -1.4 12761.5 -8.7 1404.7 9.1 14093.3 -10.2 1157.0 -26.3 escin 0,5μΜ 13008.5 19.5 1315.0 -3.8 12787.8 -8.5 1303.3 1.3 12340.6 -21.4 1344.9 -14.3 Dermo Abs 1/2000 11773.0 8.2 1480.0 8.3 10841.0 -22.4 1411.0 9.6 11058.0 -29.6 1526.0 -2.8 Dermo Abs / 3000 11207.0 3.0 1319.0 -3.5 11766.0 -15.8 1440.0 11.9 10422.0 -33.6 1520.0 -3.2 Dermo Br 1/1000 9896.0 -9.1 1422.0 4.0 10378.0 -25.7 1306.0 1.5 10894.0 -30.6 1890.0 20.4 Dermo Br 1/2000 10244.0 -5.9 1456.0 6.5 10096.0 -27.8 1390.0 8.0 10439.0 -33.5 1541.0 -1.8 Dexametha area 0.6ug / ml 10235.8 -5.9 1339.6 -2.0 11097.4 -20.6 1406.6 9.3 13095.0 -16.6 1131.0 -27.9

Substance tested unstimulated cells cells stimulated LPS lug / ml TNF alpha stimulated cells 2 ng / ml IL6 (pg / ml) % varied e IL8 (Pg / ml) % varied e IL6 (pg / ml) % vary to you IL8 (Pg / ml ) % vary to you IL6 (Pg / ml ) % vary to you IL8 (Pg / ml ) % varies to you Cell witness 2054.5 2610.0 8166.1 4819.5 8543.4 5547.2 Solvent control 2084.0 2497.5 7990.3 4626.7 8602.3 5024.3 Dermo-HDC 10μΜ 2543.6 23.8 2716.8 4.1 5295.6 -33.7 3273.8 -32.1 8037.9 -5.9 4971.6 -10.4 Dermo-HDC 15μΜ 2188.7 6.5 2328.9 -10.8 1961.2 -75.5 1206.2 -75.0 7346 TP -14.0 5026.6 -9.4 Dermo-HDC 20μΜ 2109.3 2.7 2185.1 -16.3 4013.7 -49.8 2943.8 -38.9 6262.6 -26.7 4293.5 -22.6

Ο ι 2 'Ο Ο 3 1 8 - 2 3 -08- 2012

ί Dermo-Es 2μΜ 1896.8 -7.7 2865.5 η <> i - _ z. . -13.4 4483.1 -7.0 89 · 'i, 7 4.2 5026.6 -9.4 Dermo-Es ΙμΜ 2194.8 6.8 2819.5 8.0 3550.0 -55.6 2284.9 -52.6 9113.3 6.7 5997.2 8.1 Dermo-Es 0,5μΜ 2212.8 7.7 2447.4 -6.2 5335.5 -33.2 Γ3475,2 -27.9 6639.2 -22.3 4495.0 -19.0 Dermo Abs 1/2000 1925.8 -6.3 2283.9 -12.5 5348.6 -33.1 2084.3 -56.7 6756.8 -20.9 2925.3 -47.3 Dermo Abs 1/3000 2195.6 6.9 2610.2 a, a 5558.2 -30.4 2353.6 -51.2 6314.1 26.1 2608.8 53.0 Dermo B 300 2369.4 15.3 3011.1 15.4 5746.5 -28.1 2539.2 -47.3 6808.3 -20.3 2757.5 -50.3 Dermo B 1/2000 2103.1 2.4 2699.4 3.4 5630.3 -29.5 2260.6 -53.1 6403.1 -25.1 2585.7 -53.4 Dexamethasone at 0.6ug / ml 1823.7 -11.2 1542.8 -40.9 2215, IJ -72.3 1100.2 -77.2 3218.5 -62.3 1379.9 -75.1

The results show the differentiated action of Dermo-Es and Dermo-HdC, as well as of Dermo-B and Dermo-Abs, as follows: Dermo-Es, Dermo-Abs and Dermo-B have anti-inflammatory effect especially at microvascular level ( inhibits the ICAM expression molecule characteristic of small blood vessels and pro-inflammatory cytokines IL6 and IL8), both under inflammatory conditions of bacterial origin and in non-specific systemic inflammables; Dermo-HdC acts especially at the level of large blood vessels (inhibits VCAM), only in non-specific inflammation.

These effects support the association of Dermo-Es and Dermo HdC for a generalized anti-inflammatory effect in the vasculature in the case of anti-cellulite action, the use of Dermo-HdC in anti-edematous products (non-specific vascular inflammation in large blood vessels), but associated with Dermo -B by its anti-microbial component and anti-inflammatory response to bacterial stimuli, for the prevention of possible local infections. The association between Dermo-Abs and Dermo-Es ensures a cumulative anti-inflammatory effect at the level of the dermal microvasculature, the inhibition by Dermo-Es of the pro-inflammatory cytokines IL6 and 118 released under conditions associated with a bacterial infection being complemented by the action of Dermo-Abs and in the case of non-specific pro-inflammatory stimuli.

Demonstration of the ability to reduce the vascular permeability of herbal preparations of Hedera helix and Aesculus hypocastanum.

Q-2 Ο 1 2 - ο 3 δ 1 8 - 2 3 -08- 2012

The protective action on the blood vessels is manifested by the increase of the capillary tone produced by the contraction of the smooth muscles of the blood vessels, which leads to the increase of the resistance and elasticity of the capillaries and to the decrease of the vascular permeability. Angiogenic pathologies are associated with the appearance of edema, increasing the vascular permeability for water and proteins with large molecular masses that enter the extracellular space. One of the main modulators of this process is the VEGF (vascular endothelial growth factor), with a dual role in angiogenesis and stimulation of vascular permeabilization, whose inhibition becomes the therapeutic target in different vascular diseases.

The materials and method used are: Human VEGF Flex Set (BD CBA) and Human Soluble Protein Master Buffer Kit (BD CBA). Rites use for the detection of soluble analytes particles with different fluorescence intensities. Beads that capture a certain fluorescence intensity are bound to specific antibodies for a particular soluble protein, in this case VEGF.

VEGF pg / ml (Nestimulation) % variation VEGF pg / ml (TNF alpha stimulation) % variation Cell witness 39.3 71.4 81.4 DMSO 42.9 93.4 31.0 Dermo-Es 2uM 64.9 51.2 69.2 -3.0 Dermo-It's luM 46.2 7.5 80.1 12.2 Dermo-Es 0.5uM 48.2 12.3 95.7 34.1 Dermo-HdC lOuM 44.1 2.8 84.5 18.4 Dermo-HdC 15uM 30.4 -29.1 62.8 -12.0 Dermo-HdC 20uM 19.3 -55.1 42.1 -40.9 Dexamethasone 0.6ug / ml 28.5 -27.5 48.2 -32.4

The bioactive component Dermo-Es from Aesculus hypocastanum extract does not act on the VEGF factor under inflammatory conditions (TNFα stimulation), under basal conditions even inducing its activation equivalent to blood vessel permeabilization.

In contrast, Dermo-HdC from Hedera helix even decreases the concentration of VEGF factor by 40%, acting to reduce vascular edema. Thus, it is recommended to include DermoHdC in product formulations with anti-edema and anti-inflammatory effect, complementing the action spectrum of Dermo-Es.

Demonstration of the antimicrobial activity of lappa and Centaurea cyanus

of herbal preparations from Arctium

<2 ο ¹ 2. - ο ο ;? 1 to 2 3 -08 2012

Microbial colonization is one of the essential events that keep k. the basis of the pathogenesis of many dermatological disorders, often associated with vascular inflammation. In the microbial cultures developed under aerobic conditions from pustular and nodulochristic skin lesions the following microorganisms were isolated: Staphylococcus aureus in 41% of cases, 5 '. epidermidis in 53% and Micrococcus spp. in 45%, while in anaerobic cultures S. aureus was isolated in 39% of cases, Propionibacterium acne in 33% and S. epidermidis in 21% (Hassanzaadeh et al., 2007) To highlight the antimicrobial effect 36 micro-organisms belonging to the genus Staphylococcus were chosen as model organisms, evaluating the antipathogenic potential of the Dermo-B and Dermo-Abs compounds according to the following objectives:

1. Isolation in micro culture of microorganisms from clinical specimens of skin lesions type.

2. Qualitative testing of antimicrobial activity - adapted diffusimetric method (spot technique).

3. Determination of the minimum concentration of inhibitors - the method of binary serial microdilutions

4. Evaluation of the influence on the adhesion capacity of the inert substrate - the microtiter method with the spectrophotometric evaluation of the biomass

The qualitative testing by diffusimetric method showed the presence of an inhibition of microbial growth at the level of the extract spot deposited on the seeded culture medium. The method does not allow a semi-quantitative assessment of the antimicrobial activity due to the diffusion profile unknown through the culture medium of the compounds. The minimum inhibitory concentration (CMI) for Dermo-B and Dermo-Abs was determined, based on the content of carboxylic acids (APFCs) and flavones in the analyzed extracts:

Blue PG extract: APFC: CMI = 1,562 mg / mL for 5. aureus, -CMI = 3.12 mg / mL for S. epidermidis.Flavone: CMI = 0.86 mg / mL for 5. aureus; CMI = 1,715 mg / mL for S. epidermidis

Extract in PG from broilers: APFC: CMI = 1.69 mg / mL for 5. aureus; CMI = 0.84 mg / mL for 5. epidermidis

Also, the extracts of Blueberries and Brusture show modulatory effect for virulence factors produced mainly by the secondary metabolism of the studied microorganisms (S. aureus and S. epidermidis), but also for hemolysins and lecithinase.

V 2 Ο 1 2 - Ο Ο 5 1 9 - 2 3 -08- 2012

Demonstration of photoprotective activity against UV-A and UV-B radiation of phytotherapeutic preparations of Trifolium pratense, Aesculus hypocastanum and Centaurea cyanus

UV radiation is a major source of inflammation at the skin level, so we considered it necessary to associate in the dermatocosmetic products some phytocomposites that intervene in counteracting the harmful effects of the sun exposure (Dermo-ET, Dermo-Es and Dermo-Abs).

In irradiated skin reactive oxygen species are generated that produce oxidative degradation in lipids, proteins and DNA. Mutations at the DNA level result in activation of the p53 protein, a promoter of apoptosis at the keratinocyte level, which ultimately results in the destruction of the epithelial structure. Irradiated keratinocytes release pro-inflammatory cytokines and indirectly induce MMP-1 activation in dermal fibroblasts. ILla plays a major role in the indirect propagation of these effects.

Thus, the action of Dermo-ET, Dermo-Es and Dermo Abs preparations in the pathogenesis of photo-aging has been demonstrated by screening the following cellular parameters:

• Induction of apoptosis triggered by changes in DNA level • Cellular oxidative stress - intracellular activation of reactive oxygen species • Inflammatory status: secretion of pro-inflammatory cytokines (IL6, IL8), ILl-α as an indicator of sensitization and irritability;

• VEGF (vascular endothelial growth factor) secretion - angiogenesis promoter - the key step in the restoration of degraded skin tissue and wound healing.

The centralization of the cumulative photoprotective effects of the tested compounds is presented in the tables below (compared to the positive control N actil cysteine lOmM):

^ - 2012-00313--

N-Acetyl Cysteine 65.1 20.3 8.3 6.3 289.85 220.65 1058 1208 17896 16254

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There is a differentiated effect of the compounds, each acting on other ways of propagating cell degradation and inflammation induced by UV irradiation:

Clover extract, Dermo-ET has cellular protective effect against apoptosis induced by UV radiation, stopping the inflammatory process triggered by signaling cytokines IL6 and IL8, antioxidant especially against UV-A.

The phyto-compound Dermo-Abs is especially active in the case of UV-B irradiation: anti-apoptotic, anti-irritative by inhibition of ILla (allergy signaling cytokine) and anti-angiogenic by inhibition of VEGF factor. As opposed to UV-A radiation, it has only a weak antioxidant effect.

Dermo-Es is a cellular protector against apoptosis, anti-inflammatory and anti-irritant by stopping the IL6, IL8 and ILla signaling, both in the case of UV-A and UV-B, antioxidant especially against UV-A.

The complementarity of these effects recommends their association in dermatocosmetic products to protect the exposed areas.

According to the proposed technology, 5 different types of preparations are obtained representing bioactive intermediates for the cosmetic industry, thus for:

- the selective extract called Dermo ET: the vegetable raw material Herba Trifolium pratense harvested in the phase immediately before or after flowering, is dried in a thin bed, in the dark, in hot air flow, it is ground and introduced in the extraction plant. three steps, with ethyl alcohol 50-70% for 2 hours / step in the fast extraction plant, the obtained solutions analyzed from the point of view of the content of active substances isoflavones and flavones, are subjected to the concentration operations to recover the solvent and obtain the conditioned extract in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain Dermo ET preparation as a clear greenish brown liquid, with characteristic odor, standardized in flavonoid compounds expressed in quercetin of min.0.15 g / l00 ml and in isoflavonic aglycones of min .0.23 g / l00 mL as the sum of daidzein, genistein, formononetin and biochanin A, as determined active by HPLC, namely: daidzeine min. 10 mg / 100 mL, genistein min. 20 mg / 100 mL, formononetin min. 140 mg / 100 mL and biochanin A min. 60 mg / 100 mL, obtained in a 2: 1 ratio. 1: 1 plant: final extract;

- selective extract called Dermo Cas - wild chestnut seeds harvested from unpolluted geographical areas, analyzed from the point of view of heavy metals and pesticides, dried in hot air stream, milled and introduced into the extraction plant; performs successively in three steps, with ethyl alcohol 50-70% for 2 hours / step in the fast extraction plant, the obtained solutions analyzed from the point of view of the content of active substances are subjected to the concentration operations for solvent recovery and (λ-2012 '00618-ί 3 -08- 2012 obtaining the extract conditioner ir ρ · of.' .Eng'icc gbi. ·. Ina butylene glycol, cosmetic solvents admitted to obtain the fluid preparation, standardized in proanthocyanins of min.0,1 5 g / 100 g and total triterpenic saponins expressed in the min. Stage 2 g / 100 g, obtained in a ratio of 1: 1 plant: final extract; to obtain the dry extract after the concentration step under vacuum, to recover In the case of the extraction solvent, the obtained product is subjected to selective separation with one of the appropriate solvents: n-butanol, ethyl acetate - in ratio 2: 1 the separated organic extracts are further processed by concentration in vacuo, until the dry residue is purified. by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius obtaining a non-hygroscopic yellow-brown powder, called Dermo Es, standardized in triterpenic saponins expressed in min. 70% stage, and in flavonoids obtained in 33: 1 ratio plant: final extract;

- the selective extract Dermo EH- the raw material Hedera helix folium harvested during the period May-October, is dried in a thin bed, in the dark, in hot air flow, it is ground and introduced in the extraction plant, it is extracted successively in three stages. , with ethyl alcohol 5070% for 2 hours / step in the fast extraction plant, the obtained solutions analyzed from the point of view of the content of active substances are subjected to the concentration operations for the recovery of the solvent and obtaining the conditional extract in propylene glycol, glycerine or butylene glycol, solvents cosmetically admitted to obtain the fluid preparation, standardized in carboxylic polyphenol acids expressed in caffeic acid of min.3,3 g / lOOg, and in total triterpenic saponins expressed in hederacozide C of min.3 g / 100 g, obtained in a ratio of 1: 1 -1: 2 floor; In order to obtain the dry extract after the concentration step under vacuum, for the recovery of the extraction solvent, the obtained product is subjected to a selective separation with one of the suitable solvents: n-butanol, ethyl acetate - in ratio 2: 1 the separated organic extracts are further processed by concentration in vacuo, to the dry residue which is purified by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius, obtaining a white-brown powder, called Dermo HdC, non-hygroscopic, standardized in triterpenic saponins expressed in hederacozide C of at least 60%, obtained in 20: 1 ratio plant: final extract;

- the selective extract called Dermo B- the vegetable raw material - the roots of the harvested Arctium lappa plant is washed well with water, dried in a thin bed, in the dark, in hot air stream, milled and introduced into the extraction plant, extracted successively in three steps, with 80-96% ethyl alcohol for 2 hours / step in the fast extraction plant, the obtained solutions analyzed for the identification of polyacetylene by UV spectral analysis, after which they are subjected to the concentration operations to recover the solvent and obtain the conditioned extract in ογ - '2 Ο 1 2 - Ο Ο 3 1 9 - 2 3 -08- 2012 propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain Dermo B preparation in the form of a clear brownish-red liquid, with characteristic odor, characterized by the presence of polyacetylene and standardized in carboxylic polyphenol acids expressed as caffeic acid of min.0.3 g / l 00 g ,, obtained in ratio 1: 1 plant: final extract;

- selective extract Dermo Abs - vegetable raw material - blue grass harvested immediately after flowering, it is dried in a thin bed, in the dark, in hot air stream, it is ground and introduced in the extraction plant, it is extracted successively in three stages, with ethyl alcohol 60-80% for 2 hours / step in the fast extraction plant, the obtained solutions analyzed from the point of view of the content of active substances undergo the concentration operations to recover the solvent and obtain the conditioned extract in propylene glycol, glycerine or butylene glycol, solvents cosmetically admitted to obtain the Dermo Abs preparation as a fluid extract, in glycerine, propylene glycol or butylene glycol standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0,9 g / l00 g and in flavonoid compounds routinely expressed in min.0, 5 g / l 00 g ,, obtained in ratio 1: 1-1: 2 plant: final extract;

The advantages of the invention with respect to the prior art are the following:

- The technological process applicable at the industrial level of obtaining cosmetic preparations / ingredients involves the cultivation of the plant in an ecologically certified system - for the plants that are suitable for this system - obtaining the plant raw material necessary for the selective extraction in stages and the separation of the pharmaceutical and cosmetically active components, but also the recycling. plant materials and the resources used, by obtaining 5 standardized preparations in the active principles responsible for the biological action followed and proven by specific tests, in vitro, namely:

- selective extract called Dermo ET isolated from Herba Trifolium pratense, conditioned in the form of fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in flavonoid compounds expressed in quercetin of min.0,15 g / 100 ml and in isoflavonic aglycones of min. 0.23 g / 100 mL as the sum of daidzein, genistein, formononetin and biochanin A, quantitatively determined by HPLC, namely: daidzeine min. 10 mg / 100 mL, genistein min. 20 mg / 100 mL, formononetin min. 140 mg / 100 mL and biochanin A min. 60 mg / 100 mL, obtained in ratio 2: 1-1: 1 plant: final extract;

- selective extract - isolated from Hippocastani semen, conditioned either as a fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo Cas standardized in proanthocyanins of min.0,15 g / 100 g and total triterpenic saponins expressed in min. 2 g / 100 g, obtained in a ratio of 1: 1 plant: final extract; or in the form of dry extract in the form of a non-hygroscopic yellow-brown powder, called Dermo Es, standardized in saponins ¢ -2012-00313-2 3 -08-2012

triterpenes expressed in the stage of min.70%, and in flavonoids of min. 2% obtained in 33: 1 ratio plant: final extract;

- selective extract isolated from Hedera helix folium, conditioned either in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo EH, standardized in carboxylic polyphenol acids expressed as caffeic acid of min.0,3 g / lOOg, and in saponins total triterpenes expressed in hederacozide C of min.3 g / 100 g, obtained in the ratio 1: 1-1: 2 plant: final extract, or in the form of dry extract, called Dermo-HdC, as a non-hygroscopic, standardized in triterpenic saponins expressed in hederacozide C of at least 60%, obtained in 20: 1 ratio plant: final extract;

- selectively extracted Arctium lappa root roots, with antimicrobial properties due to the presence of polyacetylene, conditioned as a fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo B, standardized in carboxylic acid polyphenol expressed in min. g / 100 g ,, obtained in ratio 1: 1 plant: final extract;

- Selective extract isolated from Centaurea Cyanus Herb conditioned in the form of fluid extract, in glycerin, propylene glycol or butylene glycol, called Dermo Abs, standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0.9 g / 100 g and in flavonoid compounds expressed in the routine of min.0,5 g / 100 g,, obtained in ratio 1: 1-1: 2 plant: final extract;

- Obtaining some phytocomposites standardized in several active components, in order to make their therapeutic applications more efficient, as follows: Dermo-ET- flavonoid compounds and isoflavonic aglycones; Dermo-Cas - proanthocyanins and triterpenic saponins; Dermo-Es-saponins triterpenic and flavonoid; Dermo-EH and Dermo-HdC - carboxylic polyphenol acids and triterpenic saponins; Dermo B - carboxylic and polyacetylene polyphenol acids; Dermo-Abs - carboxylic polyphenol acids and flavonoid compounds.

- The invention proposes to obtain, through multiple associations, both the active principles isolated from different plants between them, as well as with caffeine (known lipolytic agent), of dermatocosmetic products of the type of cosmetics with cumulated action of dermal restructuring and anti-inflammatory skin, used in the treatment of cellulite, peripheral edema and ointments. The efficiency of these herbal products has been verified on the latest generation studies / tests at the target cell level, their association based on the complementarity of the demonstrated mechanisms of action. The dermatocosmetic products are designed so that they act in concert on all the metabolic pathways involved in the pathogenesis of the respective diseases.

The process according to the invention consists in combining the obtained cosmetic ingredients, for a superior biological, cumulative effect, synergy of the active principles existing in the phytotherapeutic preparations made according to the invention, namely: plant raw materials after harvest -

V 2 Ο 1 2 - Ο Ο 3 1 a - 2 3 -00- 2012 is processed by drying and grinding and subjected to an extraction operation, as follows: To obtain the Dermo ET preparation - the vegetable raw material Herba Trifolium pratense harvested in phase immediately before or after flowering, it is dried in a thin bed, in the dark, in warm air, it is ground and introduced in the extraction plant, it is extracted successively in three steps, with ethyl alcohol 50-70% for 2 hours / step. in the fast extraction plant, in relation to the plant: extraction solvent of 1: 20-1: 30, the obtained solutions analyzed from the point of view of the content of active substances isoflavones and flavones, are subjected to the concentration operations in a ratio of 1: 5 -1: 10, to recover the solvent and then to obtain the extract, condition in propylene glycol, glycerin or butylene glycol, cosmetic solvents admitted to obtain the Dermo ET preparation as a clear greenish-brown liquid with a characteristic odor. teristically, standardized in flavonoid compounds expressed in quercetin of min.0.15 g / 100 ml and in isoflavonic aglycones of min.0.23 g / 100 mL as the sum of daidzeine, genistein, formononetine and biochanin A, quantitatively determined by HPLC and namely: daidzeine min.10 mg / 100 mL, genistein min.20 mg / 100 mL, formononetin min.140 mg / 100 mL and biochanin A min.60 mg / 100 mL, obtained in 2: 1-1: 1 ratio plant : final extract;

To obtain the Dermo Cas-Hippocastani preparation, the wild chestnut seeds harvested from unpolluted geographical areas, analyzed from the point of view of heavy metals and pesticides, are dried in hot air stream, milled and introduced into the extraction plant; the extraction operation; is carried out successively in three steps, with ethyl alcohol 50-70% for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent of 1: 10-1: 15, the obtained solutions analyzed from the point of view of the content of active substances is subjected to the concentration operations in a ratio 1: 5-1: 10 for the recovery of the solvent and then for obtaining the conditioned extract in propylene glycol, glycerin or butylene glycol, cosmetic solvents admitted, with the obtaining of the fluid preparation, standardized in proanthocyanins of min .0.15 g / 100 g and total triterpenic saponins expressed in the min. Range of 2 g / 100 g, obtained in a ratio of 1: 1 plant: final extract; In order to obtain the dry extract after the concentration step under vacuum, for the recovery of the extraction solvent, the obtained product is subjected to a selective separation with one of the suitable solvents: n-butanol, ethyl acetate - in ratio 2: 1 the separated organic extracts are further processed by concentration in vacuo, to the dry residue which is purified by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius obtaining a non-hygroscopic yellow-coffee powder, called Dermo Es, standardized in triterpenic saponins expressed in the min. 70% stage, and in the flavonoids of min. 2% obtained in 33: 1 ratio plant: final extract;

^ -2012-00318-Î 3 -08- 2012

In order to obtain the Dermo EH preparation- the raw material Hedera helix folium harvested during the period May-October, it is dried in a thin bed, in the dark, in hot air flow, it is ground and introduced in the extraction plant, it is extracted successively in three steps. , with 50-70% ethyl alcohol for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent of 1: 20-1: 30, the obtained solutions analyzed from the point of view of the content of active substances are subjected concentration operations in a ratio of 1: 10-1: 15 to recover the solvent and then to obtain the conditioned extract in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain the fluid preparation, standardized in carboxylic polyphenol acids expressed in caffeic acid by min. 0.3 g / lOOg, and in total triterpenic saponins expressed in hederacozide C of min.3 g / 100 g, obtained in the ratio 1: 1-1: 2 plant; In order to obtain the dry extract after the concentration step under vacuum, for the recovery of the extraction solvent, the obtained product is subjected to a selective separation with one of the suitable solvents: n-butanol, ethyl acetate - in ratio 2: 1 the separated organic extracts are further processed by concentration in vacuo, until the dry residue which is purified by suspension in acetone, filtration and drying at low temperature, in hot air flow: 5060 degrees Celsius obtaining a white-brown, non-hygroscopic powder, standardized in triterpenic saponins expressed in hederacozide C of at least 60%, obtained in 20: 1 ratio plant: final extract;

In order to obtain the Dermo B preparation - the vegetable raw material - the roots of the harvested Arctium lappa plant are washed well with water, dried in a thin bed, in the dark, in hot air stream, ground and introduced into the extraction plant, extracted successively in three steps, with 80-96% ethyl alcohol for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent 1: 10-1: 15, the obtained solutions analyzed for the identification of polyacetylene by UV spectral analysis, after which are subjected to dry residue concentration operations, for the recovery of the solvent and obtaining the conditioned extract in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain the Dermo B preparation in the form of a clear brown-red liquid, with characteristic odor, characterized by the presence of polyacetylene in carboxylic polyphenol acids expressed as caffeic acid of min.0,3 g / 100 g, obtained in 1: 1 ratio plant: extra final ct;

To obtain the Dermo Abs-vegetable raw material - blue grass Centaureae cyanum harvested immediately after flowering, it is dried in a thin bed, in the dark, in warm air, it is ground and introduced in the extraction plant, it is successively extracted in three steps, with 60-80% ethyl alcohol for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent 1: 15-1: 20, the solutions obtained analyzed from the point of view of

^ -2012-00618-2 3 -08- 2012

the content of active substances is subjected to: dc concentration operations for the recovery of the solvent in the ratio 1: 10-1: 20 and obtaining the conditioned extract in propylene glycol, glycerin or butylene glycol, cosmetic solvents admitted to obtain the preparation of Dermo Abs in the form of a fluid extract, in glycerin, standardized propylene glycol or butylene glycol in carboxylic polyphenol acids expressed in caffeic acid of min.0.9 g / 100 g and in flavonoid compounds routinely expressed in min.0.5 g / 100 g, obtained in a ratio of 1: 1-1: 2nd floor: final extract.

The following are examples of embodiments of the invention, which relate to the process of obtaining standardized cosmetically active extracts from plants: Trifolium pratense herba, Hedera helix folium, Aesculus hypocastanum semen, Arctium lappa radix, Centaureae cyanum herba, in the tests performed below. highlighting the specific activity and evaluating the applications in therapeutics, as well as the dermato-cosmetic products conditioned.

Embodiment Example 1. Process for obtaining active cosmetic preparations

Obtaining the preparation Dermo ET - the vegetable raw material Herba Trifolium pratense harvested in the phase immediately before or after flowering, it is dried in a thin bed, in the dark, in hot air, it is ground and introduced in the extraction plant. It is extracted successively in three stages. , with ethyl alcohol 50-70% for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent of 1: 20-1: 30, at ambient temperature, the solutions obtained analyzed from the point of view of the content of active substances isoflavones and flavones, are subjected to the concentration operations in a ratio of 1: 5-1: 10, for the recovery of the solvent and then for obtaining the conditioned extract in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain the preparation Dermo ET in the form of a clear greenish brown liquid, with characteristic odor, standardized in flavonoid compounds expressed in quercetin of min.0,15 g / 100 ml and in isoflavo aglycones no min.0.23 g / 100 mL as the sum of daidzein, genistein, formononetin and biochanin A, quantitatively determined by HPLC, namely: daidzein min. 10 mg / 100 mL, genistein min. 20 mg / 100 mL, formononetin min. 140 mg / 100 mL and biochanin A min. 60 mg / 100 mL, obtained in ratio 2: 1-1: 1 plant: final extract;

Obtaining the Dermo Cas-Hippocastani preparation resembles wild chestnut seeds harvested from unpolluted geographical areas, analyzed from the point of view of heavy metals and pesticides, is dried in hot air stream, milled and introduced into the extraction plant; performs successively in three steps, with ethyl alcohol 50-70% for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent of 1: 10-1: 15, at ambient temperature, the solutions obtained analyzed from the point in view of the content of active substances are subjected to the concentration operations in a ratio 1: 5-1: 10 for the recovery of the solvent and then for obtaining the conditioned extract in propylene glycol, glycerin or

^ - 2012-00318-2 3 -οβ- 2012 butylene glycol, cosmetically approved solvents, obtaining the fluid preparation, standardized in proanthocyanins of min.0,15 g / 100 g and total triterpenic saponins expressed in the scale of min.2 g / l00 g, obtained in ratio 1: 1 plant: final extract; In order to obtain the dry extract after the concentration step under vacuum, for the recovery of the extraction solvent, the obtained product is subjected to a selective separation with one of the suitable solvents: n-butanol, ethyl acetate - in ratio 2: 1 the separated organic extracts are further processed by concentration in vacuo, to the dry residue which is purified by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius giving a nonhygroscopic yellowish powder called Dermo Es, standardized in saponins triterpenes expressed in the stage of min.70%, and in flavonoids of minimum 2% obtained in 33: 1 ratio plant: final extract;

Obtaining the Dermo EH preparation - the vegetable raw material Hedera helix folium harvested during the period May-October, is dried in a thin bed, in the dark, in hot air, it is ground and introduced in the extraction plant, it is extracted successively in three stages, with ethyl alcohol 50-70% for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent of 1: 20-1: 30, at ambient temperature the obtained solutions analyzed from the point of view of the content of active substances are subjected to the concentration operations in a ratio of 1: 10-1: 15 to recover the solvent and then to obtain the conditioned extract in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain the fluid preparation, standardized in polyphenol carboxylic acids expressed in caffeic acid min.0.3 g / lOOg, and in total triterpenic saponins expressed in hederacozide C of min.3 g / 100 g, obtained in the ratio 1: 1-1: 2 plant; In order to obtain the dry extract after the concentration step under vacuum, for the recovery of the extraction solvent, the obtained product is subjected to a selective separation with one of the suitable solvents: n-butanol, ethyl acetate - in ratio 2: 1 the separated organic extracts are further processed by concentration in vacuo, until the dry residue which is purified by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius obtaining a white-brown, non-hygroscopic powder, standardized in triterpenic saponins expressed in hederacozide C of at least 60%, obtained in ratio 20: 1 plant: final extract;

Obtaining the Dermo B preparation - the vegetable raw material - the roots of the harvested Arctium lappa plant is washed well with water, dried in a thin bed, in the dark, in hot air stream, ground and introduced into the extraction plant, extracted successively in three steps, with 80-96% ethyl alcohol for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent 1: 10-1: 15, at ambient temperature the obtained solutions analyzed for the identification of polyacetylene by UV spectral analysis, then subjected to concentration operations at γτ t

V 2 Ο 1 2 ~ Ο Ο 3 1 8 - 2 3 -08- 2012

dry residue, to recover the solvent and obtain the extract, condition in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted to obtain Dermo B preparation in the form of a clear reddish-brown liquid, with characteristic odor, characterized by the presence of polyacetylene and standardized in polyphenyl carboxylic acids in caffeic acid of min.3,3 g / 100 g ,, obtained in ratio 1: 1 plant: final extract;

Obtaining the preparation Dermo Abs-the vegetable raw material - blue grass Centaureae cyanum harvested immediately after flowering, it is dried in a thin bed, in the dark, in hot air, it is ground and introduced in the extraction plant, it is extracted successively in three steps. , with 60-80% ethyl alcohol for 2 hours / step in the fast extraction plant, in relation to the plant: extraction solvent 1: 15-1: 20, at ambient temperature the obtained solutions analyzed from the point of view of the content of active substances are subjected to the concentration operations for the recovery of the solvent in the ratio 1: 10-1: 20 and obtaining the conditioned extract in propylene glycol, glycerine or butylene glycol, cosmetic solvents admitted with the preparation of the Dermo Abs preparation as a fluid extract, in glycerine, propylene glycol or butylene glycol standardized in carboxylic polyphenol acids expressed in caffeic acid of min.0,9 g / 100 g and in flavonoid compounds routinely expressed of min.0,5 g / 1 00 g,, obtained in ratio 1: 1-1: 2 plant: final extract;

The embodiment example no. 2: Dermatocosmetic product, containing as biologically active substances the herbal product Dermo-ET 5.00-5-12.00%, the herbal product Dermo-HdC 0.50-5-5.00% (or Dermo-EH in the amount corresponding to the active dose of Dermo HdC), the herbal product Dermo-Es 0.10 ^ 0.70% (or Dermo-Cas in the amount corresponding to the active dose of Dermo Es) in combination with caffeine, conditioned in the form of gel or cream, usable in anti-cellulite therapy.

Conditioning formula (anti-cellulite cream):

Nr. crt. INCI Quantitative formula 1. Aqua Ad 100 2. Mineral Oii 3.00x15.00 3. Glyceryl Stearate 0.30x3.00 4. lanolin 1.00x5.00 .. ..

t \ - 2 O 1 1 - O O 5 1 8 - 2 3 -OB- 2012

5. Petrolatum 2.00-8.00 6. Glycerin 3.00-5.00 7. Isopropyl targets state 1.00-5.00 8. Potassium Cetyl Phosphate 1.00-5.00 9. Dimethyl isosorbide 1.00-5.50 10. Cetyl Alcohol 1.00-5.00 11. Hedera Helix Extract 0.50-5.00 12. Aesculus Hippocastanum Extract 0.10-0.70 13. Trifolium pretense extract 5.00-12.00 14. Propylene Glycol (and) Diazolidinyl Urea (and) Methylparaben (and) Propylparaben 0.50-1.00 15. Sodium Hydroxide 0.15-0.40 16. carbomer 0.15-0.40 17. dimethicone 1.00-3.00 18. Tocopherol acetate 0.10-0.50 19. Perfume 0.10-0.30 20. BHA 0.02-0.20 21. Disodium EDTA 0.05-0.20 22. Caffeine 0.10-0.50

Conditioning formula (anti-cellulite gel):

όκ- 2 0 Ί 2 - 0 0 3 1 3 - 2 3 -D8- 2012

1. Aqua Ad.100 2. Hedera Helix Extract 0.50 = 5.00 3. Aesculus Hippocastanum Extract 0.10 = 0.70 4. Dimethyl isosorbide 1.00 = 5.50 5. PEG-12 1.00 = 10.00 6. Trifolium pretense extract 5.00 = 12.00 7. PPG-26-Buteth-26 (and) PEG-40 Hydrogenated Castor Oii 0.50 = 5.00 8. carbomer 0.10 = 1.50 9. Sodium Hydroxide 0.10 = 1.50 10. Diazolidinyl Urea (and) Methylparaben (and) Propylparaben (and) Propylene Glycol 0.50 = 1.00 11. Perfume 0.01 = 0.20 12. EDTA 0.05 = 0.20 13. Caffeine 0.10 = 0.50 Proc the dermatocosmetic door will be conditioned in two variants intended for topical application, in

depending on the appropriate treatment stage: massage cream, respectively gel for a quick penetration of the active principles when applied at home. By combining biologically active phytocomposites with complementary effects, the product exhibits anti-inflammatory and vascular permeability reduction (Dermo-HdC and Dermo-Es), reconsolidation of the extracellular protein matrix with supportive role and acceleration of fibroblast cell turn-over, cumulative. with the antioxidant and photoprotective effect against UVA and UV-B radiation, including stopping angiogenesis (cumulative effects of Dermo-ET compound), as well as inhibiting caffeine-induced lipogenesis, thus making a concerted attack on all metabolic pathways of progression. of cellulite.

The embodiment example no. 3: Dermatocosmetic product, containing as biologically active substances the herbal product Dermo-ET 5.00 = 12.00%, the herbal product Dermo-HdC 1.00 = 2.00% (or Dermo-EH in the amount corresponding to the active dose of Dermo HdC),

Λ - 2 Ο 1 2 - Ο Ο 3 ί 8 - 2 3 -08- 2012 Dermo-B herbal product 1.00-5.00%, conditioned in the form of gel or cream, usable in alternative therapies for peripheral vascular edema.

Conditioning formula (anti-edema gel):

Crt. INCI Quantitative formula 1. Aqua Ad.100 2. glycerin 3.00-5.00 3. Hedera Helix Extract 1.00-2.00 4. Trifolium pretense extract 5.00-12.00 5. Radix bardanae extract 1.00-5.00 6. Dimethylisosorbide 0.50-1.00 7. Diazolidinyl Urea (and) Methylparaben (and) Propylparaben (and) Propylene Glycol 0.50-1.00 8. carbomer 0.50-1.50 9. Sodium Hydroxide 0.50-1.50 10. Pentylene glycol 3.00-5.00 11. Perfume 0.15-0.25 12. EDTA 0.05-0.10

Conditioning formula (cream with anti-edema effect):

No. No. QUANTITATIVE FORMULA INCI w / w% 1. Aqua Ad 100 2. Mineral Oii 1.00-10.00

^ 2012-03313-2 3-08-2012

3. Ceteareth-15 (and) Glyceryl Stearate 0.50-5.50 4. Petrolatum 2.00-7.00 5. Cetyl Alcohol 1.00-4.00 6. Glyceryl Stearate 0.50-4.50 7. Methyl Glucose Sesquistearate 0.7-3.20 8. lanolin 1.00-4.00 9. Propylene Glycol (and) Diazolidinyl Urea (and) Methylparaben (and) Propylparaben 0.50-1.00 10. Potassium Cetyl Phosphate 0.25-2.50 11. Dimethylisosorbide 0.50-2.00 12. Polidimethylsiloxane 0.1-0.50 13. Hedera Helix Extract 1.00-2.00 14. Trifolium pretense extract 5.00-12.00 15. Radix bardanae extract 1.00-5.00 16. Perfume 0.05-0.15 17. carbomer 0.10-0.30 18. Sodium Hydroxide 0.10-0.30 19. Tocopheryl Acetate 0.10-0.50 20. BHA 0.05-0.10 'The dermatocosmetic product accumulates the anti-inflammatory and decrease action of

the vascular permeability of Dermo-HdC, with the antimicrobial, antioxidant and anti-inflammatory effects of Dermo-B, and with the effect of dermal restructuring through the synthesis of structural proteins and accelerating the rate of cell proliferation induced by Dermo-ET, being recommended especially for the attenuation of the former forms of peripheral vascular edema, in the form of cream \

^ -2012-00318-2 3 -08- 2012

The embodiment example no. 4: Dermatocosmetic product, containing biologically active substances the Dermo-ET 0.10-2.00% titer, the Dermo-Es phytotherapeutic product 0.03-0.20% (or Dermo-Cas in the amount corresponding to the active Dermo-Es dose), the Dermo-Abs 0.10 phytotherapeutic product -2.50%, conditioned in the form of a serum or cream, usable in the treatment of periorbital hyperchromia (circumnavigation).

Conditioning formula (antiretroviral serum):

Crt. INCI Quantitative formula 1. Aqua Ad.100 2. Hydroxyethyl Acrylate / Sodium Acryloyldimethyl Taurate Copolymer 0.10-0.90 3. Isononyl isononanoate 5.00-12.00 4. Xylitylglucoside 3.00-6.00 5. EDTA 0.10-0.50 6. Phenethyl alcohol and Caprylyl glycol 0.60-1.00 7. Pentylene glycol 1.00-5.00 8. Trifolium pretense extract 0.10-2.00 9. Aesculus Hippocastanum Extract 0.03-0.20 10. Centaur cyanus extract 0.10-2.50 11. Caffeine 0.10-1.5

Conditioning formula (eye contour cream with anti-rainbow effect):

Crt. INCI Quantitative formula 1. Aqua Ad.100 2. Polyglyceryl-3 Methylglucose Distearate 1.00-3.00 3. Glyceryl Stearate 1.00-4.00

Α-2 Ο 1 2 - Ο ί) 6 ι 8 - 2 3 -ΟΒ- 2012

4. Stearyl Alcohol 1.00-3.00 5. Dimethylisosorbide 1.00-5.00 6. Cyclopentasiloxane (and) dimethicone crosspolymer 1.00-3.00 7. Squalan olives 1.00 + 4.00 8. bisabolol 0.10-0.20 9. Trifolium pretense extract 0.10 + 2.00 10. Aesculus Hippocastanum Extract 0.03 + 0.20 11. Centaur cyanus extract 0.10 + 2.50 12. carbomer 0.10 + 0.50 13. Sodium Hydroxide 0.15 + 0.50 14. Perfume 0.05 + 0.20 15. Phenoxyethanol (and) Potassium sorbate 0.50 + 1.00 16. Disodium EDTA 0.05 + 0.20 17. Caffeine 0.10 + 1.5

Due to the association of biologically active phytocomposites with complementary and cumulative effects, the dermatocosmetic product presents a dermo-restitutive action through the activity of Dermo-ET (stimulation of cell proliferation rate, collagen synthesis by inhibition of degradative enzymes and expression of integrins responsible for tissue tightness), - inflammatory and antioxidant (Dermo-Abs and Dermo-Es), photoprotective, including inhibition of angiogenesis triggered by UV radiation (Dermo-Abs, Dermo-Es and Dermo-ET), and anti-irritant (Dermo-Abs by cytokine inhibition ILla), being especially recommended for the treatment of periocular hyperchromia (ointment), in the form of cream or serum.

Claims (9)

1. Selective extract called Dermo ET characterized in that it is isolated from Trifolii grass, conditioned as a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in flavonoid compounds expressed in quercetin of min.0.15 g / 100 ml and in isoflavonic aglycones of min.0.23 g / 100 mL as the sum of daidzein, genistein, formononetin and biochanin A, determined quantitatively by HPLC, namely: daidzeine min. 10 mg / 100 mL, genistein min. 20 mg / 100 mL, formononetin min. 140 mg / 100 mL and biochanin A min. 60 mg / 100 mL, obtained in ratio 2: 1-1: 1 plant: final extract; 2. Selective extract called DermoCas characterized in that it is isolated from semen Hippocastans, conditioned either in the form of fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in proanthocyanins of min. 0.15 g / 100 g and total triterpenic saponins expressed in escina de min.2 g / 100 g, obtained in ratio 1: 1 plant: final extract; or in the form of a dry extract in the form of a non-hygroscopic yellow-brown powder, called Dermo Es, standardized in triterpenic saponins expressed in min.70% scale, and in flavonoids of min 2% obtained in 33: 1 ratio plant: final extract; 3. Selective extract called Dermo EH characterized in that it is isolated from Folium Hederae Helicis, conditioned either in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in carboxylic polyphenol acids expressed as caffeic acid of min.3,3 g / lOOg, and in total triterpenic saponins expressed in hederacozide C of min.3 g / 100 g, obtained in a ratio of 1: 1-1: 2 plant: final extract, or in the form of dry extract as a white-coffee powder, non-hygroscopic, called Dermo HdC, standardized in triterpenic saponins expressed in C hederacozide of min.60%, obtained in 20: 1 ratio plant: final extract; 4. Selective extract called Dermo B, characterized in that it is isolated from Radix Bardanae, with antimicrobial properties due to the presence of polyacetylene, conditioned in the form of a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in polyphenol carboxylic acids expressed in caffeic acid. 0,3 g / 100 g,, obtained in a ratio of 1: 1 plant: final extract; 5. Selective extract called Dermo Abs, isolated from Cyani Herb conditioned as a fluid extract, in glycerin, propylene glycol or butylene glycol, standardized in polyphenol acids tf-7012'00618-2 3-0B-2012 carboxylic acids expressed in caffeic acid by min. 0.9 g / 100 g and in flavonoid compounds. routinely expressed as min. 0.5 g / 100 g, obtained in ratio 1: 1-1: 2 plant: final extract; 6. Process for obtaining the dermato-cosmetic phytotherapeutic products defined in claims 1-5, characterized in that the technology exploits at industrial level the therapeutic potential of the plant raw materials which, after harvesting, is dried and subjected to a common primary extraction operation for all materials. bonuses made with ethyl alcohol of different concentrations depending on the component sought, ranging from 40-96%; the obtained extracts are still subjected to processing by bleaching with activated carbon, vacuum concentration and conditioning in the appropriate cosmetic solvent, in the established ratio obtaining selective fluid extracts; In the case of dry extracts, after the concentration step under vacuum, the product obtained is subjected to a selective separation with one of the suitable solvents: nbutanol, ethyl acetate - the separate organic extracts are further processed by vacuum concentration, until the dry residue is purified. by suspension in acetone, filtration and drying at low temperature, in hot air flow: 50-60 degrees Celsius; the standardized extracts thus obtained are subjected to chemical analyzes to determine the concentrations of active substances and the traces of solvents used are each conditioned by the purpose of their intended use, becoming pharmaceutically or cosmetically active intermediates. 7. Dermatocosmetic products of the anti-cellulite type, containing as biologically active substances the phytotherapeutic products Dermo-ET, Dermo-HdC (or Dermo-EH), Dermo-Es (or Dermo-Cas), associated with caffeine, as defined in claim 1-3, conditioned. in the form of a cream or gel, containing 5.00 + 12.00% Dermo-ET, 1.00 + 2.00% Dermo-HdC (or Dermo-EH in the amount corresponding to the active dose of Dermo HdC), 0.10 + 0.70% Dermo-Es (or Dermo- Case in the amount corresponding to the active dose of Dermo Es), being applicable in the therapy of lipodystrophic disorders. 8. Dermatocosmetic anti-dandruff type products, containing biologically active substances Dermo-ET, Dermo-B, Dermo-HdC (or Dermo-EH) herbal products, as defined in claim 1-4, conditioned in the form of a cream or gel, containing 5.00 + 12.00% DermoET, 1.00 + 5.00% Dermo-B, 1.00 + 2.00% Dermo-HdC (or Dermo-EH in the amount corresponding to the active dose of Dermo HdC), being applicable in the combination therapy of peripheral vascular edema. 9. Dermatocosmetic products of the anti-arcane type, containing as active biological substances the phytotherapeutic products Dermo-ET, Dermo-Abs, Dermo-Es (or Dermo-Cas), defined in ^ -2012-06618-2 3 -0Β-2012 claim 1- 5, conditioned in the form of cream or serum, containing 0.10 ^ -2.00% DermoET, 0.10 ^ -2.50% Dermo-Abs, Dermo-Es 0.03-M) .20% (or Dermo-Cas in the amount corresponding to the active dose of Dermo Es), being applicable in the therapy of periorbital hyperchromia (circons).