PL233572B1 - Method for obtaining skin treatment and care preparation and the skin treatment and care preparation - Google Patents

Description

Description of the invention

The subject of the invention is a method of obtaining a skin care preparation from extracts from the root of Echium italicum L., which are a source of biologically active substances such as: allantoin, shiconin or polyphenols. The preparation obtained by the method according to the invention is used for skin care after wounds, burns of various etiologies, bacterial and fungal infections.

It is known to use allantoin as an active ingredient in various types of dermatological and skin care preparations in the form of ointments or powders for the care of the skin of both adults and children, and even infants.

The use of allantoin in cosmetics is also known, for example for the production of moisturizing and softening the skin of liquid emulsions, creams, shower gels, antiperspirants, as well as care cosmetics for children. According to Fedoreyev et al. In the article "Production of allantoin, rabdosiin and rosmarinic acid in callus cultures of the seacoastal plant Mertensia maritima (Boraginaceae)" (Plant Cell Tiss. Organ. Cult., 2012, 110: 183-188), allantoin found use in over 1,300 cosmetic preparations.

Allantoin, used in most of the preparations available on the market, is obtained by chemical synthesis. It is a relatively complicated process. In turn, known methods of isolating allantoin from extracts of plants commonly found in Poland, such as comfrey, are limited to obtaining only allantoin as the main active substance found in this raw material, hence preparations containing allantoin derived from such sources, e.g. comfrey ointment or gels comfrey, often without other components with a healing effect.

Another active substance of plant origin that is essential for the proper functioning of the skin's repair mechanisms is shikonin.

As reported, among others Papageorgiou et al., In the article "The chemistry and biology of alkannin, shikonin, andrelated naphthazarin naturalproducts (Angew. Chem. Int. Ed., 1999, 38: 270-300); Ge et al., In the article "Effects ofrare earth elements on the growth of Arnebia euchroma cells and the biosynthesis of shikonin (Plant Growth Regul., 2006, 48: 283-290); Chen et al., In the article "Cellular pharmacology studies of shikonin derivatives (Phytother. Res., 2002, 16: 199-209) or Zare et al., In the article" Bioprocess engineering of Echium italicum L .: induction of shikonin and alkane derivativers by twoliquid-phase suspension cultures (Plant Cell Tiss. Organ. Cult., 2010, 100: 157-164), shiconin exhibits potent anti-inflammatory, antitumor, antithrombotic and antibacterial activity, especially against gram-positive bacteria.

Teresa Bujak in the article entitled "Aloe ointments with Chinese herbal extracts" (Pol. J. Cosmetol. 2014, 17 (3): 231-237) describes that shikonin has strong antibacterial properties, and the root of red-root recurrence, which is rich in this compound, is used by medicine as a medicine for skin eczema.

Yoo et al., In the article "Lithospermum erythrorhizon extractprotects keratinocytes and fibroblasts against oxidative stress (J. Med. Food, 2014, 00 (0): 1-8), showed that the recurrence extract, rich in shiconin, had a protective effect on human cells skin, protecting it against oxidative stress caused by hydrogen peroxide and, consequently, delaying the aging process.

Another plant source for obtaining shikonin is Alcanna tinctoria root extract. It should be emphasized, however, that on an industrial scale, shikonin is obtained by biotechnological methods, and obtaining metabolites in in vitro cultures causes relatively high costs of such a process.

Another important component of dermatological preparations or skin care cosmetics are vitamins, especially vitamin F, which is a mixture of unsaturated linolenic fatty acids, including in the composition of vegetable oils. According to Ando et al., In the article "Linolenic acid and α-linolenic acid lightens ultraviolet-induced hyperpigmentation of the skin (Arch. Dermatol. Res., 1998, 290 (7): 375-81), these acids play in the body a very important physiological role in the process of regeneration of the epithelium and connective tissue of the skin, which directly affects the maintenance of the skin in good condition. It has also been shown that vitamin F deficiency leads to various types of skin changes such as excessive peeling of the epidermis, dry skin, seborrhea or acne-prone skin.

PL 233 572 B1

The aim of the invention was to develop a simple, ecological and economical method of obtaining biologically active substances, mainly allantoin and shiconin, enriched with polyphenolic antioxidants from one easily available natural raw material, used to develop a simple composition of the preparation with high efficiency in the regeneration and care of damaged skin.

The method of obtaining a skin care preparation using the root of Echium italicum L. to obtain extracts containing allantoin and shiconin with a mixture of polyphenols, according to the invention, is characterized by the fact that it is previously cleaned, separated from the bark, dried, ground and sieved through a sieve with a diameter of mesh size not larger than 1.6 mm, the root of Echium italicum L., is moistened with ethyl alcohol and after the alcohol has evaporated, it is poured with a polar solvent in the form of distilled water in a root / water weight ratio of 1 to 4, shaken for at least 4 hours, and then is extracted in the cavitation process using an ultrasonic bath for 30-60 minutes at 35-45 ° C, repeating the process several times and after centrifuging the solids, an extract is obtained with a content of 0.25 to 0.35 g of allantoin in 100 ml of solution . Additionally, the extract contains a mixture of polyphenols whose content ranges from 75 to 120 mg of gallic acid equivalent per 100 ml of extract.

The bark separated from the root of Echium italicum L., prepared as the root, is poured over with cold-pressed linseed oil in a weight ratio of root bark / oil of 1 to 10 and shaken continuously for 48-72 hours, then the solids of the mixture are filtered off, obtaining extract with a content of shikonin in the amount of 0.2 to 0.3 g per 100 ml of oil extract with a set of valuable unsaturated fatty acids and vitamins derived from the oil. In the final stage, the aqueous extract containing allantoin and polyphenols is emulsified with a lipophilic ointment base, preferably with eucerin, in a weight ratio of 1: 1, continuously grinding in a porcelain mortar heated in a 60-75 ° C water bath, and after obtaining a homogeneous form, added the oil extract of shikonin in the weight ratio to the previous ingredients is reduced by half, and after obtaining a homogeneous liniment, it is cooled to 8-7 ° C, obtaining the form of a solidified ointment.

The preparation obtained by the method according to the invention, based on a lipophilic ointment with a mixture of biologically active substances such as allantoin, shiconin, or polyphenols derived from the root of Echium italicum L., as well as vitamins included in the linseed oil according to the invention, is characterized by the fact that components such as ointment; water extract of Echiumitalicum root L. with an allantoin content in the range from 0.25 to 0.35 g per 100 g of extract with a mixture of polyphenols in the amount of 75 to 120 mg expressed as gallic acid equivalent in 100 g of extract and oil extract with a shiconin content in the range of 0.2 up to 0.3 g per 100 g of extract along with a set of unsaturated fatty acids and vitamins, in weight proportions as 1: 1: 0.5.

The advantage of the invention is a simple, ecological and economical method of obtaining with the use of two types of extraction root - water and root bark - linseed oil, two biologically active substances - allantoin and shiconin along with valuable polyphenols with antioxidant properties from one easily available natural raw material containing optimally high the content of the substances mentioned.

The preparation according to the invention has a simple composition, with the content of allantoin and shiconin active substances, additionally supported by antioxidants and linseed oil components, such as unsaturated fatty acids and vitamins, mainly vitamin F. The conducted research has shown the high effectiveness of the preparation in skin care, and at the same time lack of its toxic properties. .

The invention is illustrated in the following examples.

P r z k ł a d 1

300 g of previously cleaned, separated from the bark, dried, ground and sieved through a sieve with a mesh diameter of 1.6 mm of the root of Echium italicum L., wetted with ethyl alcohol and, after evaporation of the alcohol, poured over 1200 ml of distilled water and shaken for at least 4 hours, after In the cavitation process, using an ultrasonic bath with ultrasound power of 240 W and frequency of 35 kHz, extraction was carried out for 30 minutes at a temperature of 45 ° C, repeating the process several times. Then, the solids of the mixture were separated from the solution by filtering the mixture through sterile gauze. With the use of capillary electrophoresis against the allantoin standard of> 98% purity, the allantoin content was determined at the level of 0.25 g in 100 g of aqueous extract. Additionally, the total content of polyphenolic compounds was determined using the Folin-Cicalteau method at the level of 75 mg of gallic acid equivalent per 100 g of extract.

PL 233 572 B1

In the second stage, 50 g of the bark separated from the root of Echium italicum L., previously dried, ground and sieved through a sieve with a mesh diameter of 1.5 mm, was moistened with ethyl alcohol and, after alcohol evaporation, 500 ml of cold pressed linseed oil was poured and shaken continuously for 48 hours, then the solids of the mixture were centrifuged at 5000 rpm for 10 minutes.

The obtained supernatant was additionally filtered through a sterile gauze by pouring the oil extract through several times folded and placed in a funnel sterile gauze. In the obtained extract by spectrophotometric method at the wavelength of 530 nm in relation to the standard of> 98% purity, the content of shikonin was determined in the amount of 0.3 g per 100 g of extract.

In the final stage, 200 ml of the aqueous extract containing allantoin were emulsified with 200 g of eucerin, continuously grinding in a porcelain mortar heated in a water bath to 75 ° C, and after obtaining a homogeneous form, 100 g of oil extract was added, and then, after obtaining a homogeneous liniment, they were cooled to temperature 7 ° C, obtaining the form of a solidified ointment with a cherry-red color.

P r z k ł a d 2

200 g of previously cleaned, separated from the bark, dried, ground and sieved through a sieve with a mesh diameter of 1.5 mm of the root of Echium italicum L., wetted with ethyl alcohol and, after evaporation of the alcohol, poured over 800 ml of distilled water and shaken for at least 4 hours, In the process of cavitation with the use of ultrasonic bath with ultrasonic power of 240 W and frequency of 35 kHz, extraction was carried out at 35 ° C for 60 minutes, repeating the process several times. Subsequently, the solids of the mixture were centrifuged at 5000 rpm for 10 minutes, after which, using capillary electrophoresis against an allantoin standard of> 98% purity, the allantoin content was determined to be 0.35 g in 100 g of aqueous extract. Additionally, the total content of polyphenolic compounds was determined by the Folin-Cicalteau method at the level of 120 mg of gallic acid equivalent per 100 g of extract.

In the second stage, 100 g of the bark separated from the root of Echium italicum L., previously dried, ground and sieved through a sieve with a mesh diameter of 1.6 mm, was moistened with ethyl alcohol and, after the alcohol had evaporated, 1000 ml of cold pressed linseed oil were poured and shaken continuously for 72 hours. hours. The solids of the mixture were filtered off with sterile gauze, and the obtained supernatant was additionally filtered through several folded sterile gauze. In the obtained extract, the content of shikonin was determined by the spectrophotometric method at a wavelength of 530 nm with reference to the shiconin standard of> 98% purity, in the amount of 0.2 g per 100 g of extract.

In the final stage, 100 g of the aqueous extract containing allantoin was emulsified with 100 g of eucerin, continuously grinding in a porcelain mortar heated in a water bath to 60 ° C, and after obtaining a homogeneous form, 50 g of oil extract was added and cooled to 8 ° C, obtaining the form of a solidified ointment with a cherry-red color.

P r z k ł a d 3

Composition of the skin care preparation:

- 50 g of lipophilic ointment based on eucerin,

- 50 g of an aqueous extract of the roots of E. italicum L. with an allantoin content of 0.35 g / 100 g of extract and a polyphenol content of 75 mg of gallic acid equivalent per 100 g of extract,

- 25 g of oil extract of the root bark of E. italicum L. with a shiconin content of 0.2 g / 100 g with a set of valuable unsaturated fatty acids and vitamins.

P r z k ł a d 4

Composition of the skin care preparation:

- 250 g of lipophilic ointment based on eucerin,

- 250 g of an aqueous extract of the roots of E. italicum L. with an allantoin content of 0.25 g / 100 g of extract and a polyphenol content of 120 mg of gallic acid equivalent per 100 g of extract,

- 125 g of E. italicum L. root bark oil extract with a shiconin content of 0.3 g / 100 g of extract with a set of valuable unsaturated fatty acids and vitamins.

In the next example, the effect of the preparation on the viability of skin cells, expressed by the activity of succinate dehydrogenase and the stability of cell membranes using human dermal fibroblasts, was investigated.

Example 5 g of the preparation according to the invention was dissolved in 5 ml of a 1: 1 water-ethanol solution using an ultrasonic bath with a power of 240 W and a frequency of 35 kHz heated to a temperature of 35 ° C. Several solutions with the following concentrations were prepared from the obtained solution: 25, 50, 75, 100, 125 and

PL 233 572 Β1

150 µg / ml, which was used for cell culture studies and analyzes by spectrophotometric methods, such as MTT based on the evaluation of the succinate dehydrogenase activity of living cells and NR on the stability of cell membranes.

The effect of the preparation of various concentrations on the activity of succinate dehydrogenase in living cells of human dermal fibroblasts and the stability of their membranes is presented in Table 1.

Table 1

Concentration (μ ^ / ηιΐ) Method MTT NO Results k in% Control 100 ± 0.79 100 ± 3.66 25 102.76 ± 1.15 97.56 ± 1.84 50 102.95 ± 0.57 101.52 ± 2.70 75 103.54 ± 0.76 100.00 ± 4.88 100 104.33 ± 3.96 100.91 ± 1.51 150 105.12 ± 2.43 100.30 ± 2.43

The presented results indicate that with the increase in the concentration of the preparation, the activity of succinate dehydrogenase increases, which reflects the positive effect of the preparation on increasing the viability of cells of the tested fibroblasts, which indirectly proves the care properties of the preparation in the regeneration of damaged skin.

The results of the NR test do not show any effect of the preparation on the stability of cell membranes, which confirms the absence of toxic properties of the preparation.

Additionally, in the example showing no toxicity of the preparation, the observation of human fibroblast cells under a microscope was performed using the May-Grunwald-Giemsa method, in which the stained cell nuclei are dark blue in color and the cytoplasm in red-violet color.

The microscopic images are shown in the figure, which shows healthy human fibroblast cells stained with the May-Grunwald-Giems method, the darker areas are the nuclei and the lighter ones around them are the cytoplasm.

Fig. 1 shows a control image - without the use of the formulation according to the invention, and Fig. 2 and Fig. 3 - human fibroblast cells treated with a formulation solution of 25 µg / ml and 75 µg / ml, respectively. Visualization of the effect of the preparation on healthy human fibroblast cells did not show any significant differences in the morphology of these cells compared to the control.

Claims (2)

Patent claims 1. The method of obtaining a skin care preparation using the root of the Italian Echium italicum L. to obtain extracts of allantoin and shiconin with polyphenols, characterized in that the previously cleaned root of Echium italicum L. is separated from the bark, dried, ground, sieved through a sieve with a mesh diameter of not more than 1.6 and wetted with ethyl alcohol, and after the alcohol has evaporated, it is poured with a polar solvent in the form of distilled water in a root / water weight ratio of 1 to 4, shaken for at least 4 hours, and then extracted by cavitation with the use of an ultrasonic bath for 30-60 minutes at a temperature of 35-45 ° C, repeating the process several times and after centrifuging the solids, an extract is obtained with a content of 0.25 to 0.35 g of allantoin in 100 g of solution, additionally the extract contains polyphenols the content of which is between 75 and 120 mg The amount of gallic acid equivalent per 100 g of extract, while the bark separated from the root of Echium italicum L., prepared as the root, is poured with cold-pressed linseed oil in a weight ratio of root bark / oil of 1 to 10 and shaken continuously for 48 -72 hours, then the solids of the mixture are filtered to obtain an oil extract with a shiconin content in the amount of 0.2 to 0.3 g per 100 g of extract enriched with a set of unsaturated fatty acids and vitamins derived from the oil, the final In this stage, the aqueous extract containing allantoin and polyphenols is emulsified with a lipophilic ointment base, preferably with eucerin, in a weight ratio of 1: 1, constantly grinding in a porcelain mortar heated in a water bath to 60-75 ° C, and after obtaining a homogeneous form, add Shikonin oil extract in the weight ratio to the previous ingredients by half as much, and after obtaining a homogeneous liniment, it cools to 8-7 ° C, obtaining the form of solidified ointment. 2. Preparation for skin care, based on lipophilic ointment in the form of eucerin, which is a homogeneous mixture of biologically active substances such as allantoin, shikonin or polyphenols derived from the root of Echium italicum L. and a set of unsaturated fatty acids and vitamins contained in cold pressed linseed oil, characterized by ingredients such as an ointment base; water extract from the root of Echium italicum L. with an allantoin content in the range from 0.25 to 0.35 g per 100 g of extract together with a mixture of polyphenols in the amount of 75 to 120 mg expressed as gallic acid equivalent in 100 g of extract and oil extract from the content of shikonin in the range from 0.2 to 0.3 g per 100 g of extract together with a set of unsaturated fatty acids and vitamins, in weight proportions as 1: 1: 0.5.