Medication, possessing wound-healing activity

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine, in particular to pharmacology, to medication, possessing wound-healing activity. Application of hipaconitine as medication, possessing wound-healing activity is disclosed.

EFFECT: hipactonitin possesses expressed wound-healing activity.

6 tbl, 1 ex

 

The invention relates to medicine, specifically to pharmacology and cellular technologies.

There are a large amount of money, having wound healing activity: Metrorail, solcoseryl, sea buckthorn oil, sodium nukleinat and others [1, 2].

The disadvantage of these tools is often low efficiency [2].

The problem solved by the present invention, is an expansion of the means, with pronounced wound healing activity.

This object is achieved by the use of hypaconitine as a means of having wound healing activity.

New in the present invention is the use of hypaconitine as a means of having wound healing activity.

Used the original tool hypaconitine developed and received NII pharmacology" SB RAMS (, Tomsk, Russia) in cooperation with the National research Irkutsk State Technical University, Irkutsk) and represented 0,00025% aqueous solution of this alkaloid. Hypaconitine were extracted from the grass plants in the family Ranunculaceae in the form of free bases by standard extraction method [3].

An important role in tissue regeneration belongs resident progenitor, including stem (SC), cells [4, 5]. However, there are pharmacological strategy p is generative medicine, which consists in stimulation of endogenous parental cells [5]. Moreover, activation of the latter may arise from the direct effects of pharmacological agents on progenitor elements, and the result of stimulation of the cells of the tissue microenvironment, indirectly determining the accelerated course of reparative processes [5, 6].

Earlier authors was shown to accelerate the healing of skin wounds in complex extracts and alkaloid fraction of iokasti high [7]. Maximum healing effect was observed when using the sum of alkaloids, among which the dominant is Latin [8]. At the same time using the method of thin-layer chromatography in the alkaloid fraction was set to the content of other alkaloids, including hypaconitine. Hypaconitine (C33H45NO10),

is a heterocyclic nitrogen-containing organic compound of plant origin. Often it is extracted from plants of the genus Aconite, although it occurs in other plants [7, 9]. It is known that hypoacidity, as well as some other alkaloids aconitine group, has analgesic activity [10]. The ability of hypaconitine to stimulate regeneration processes, including wound healing, are not described in literature. The experimental is t showed unexpected results.

The fact of the use of Wapakoneta with achieving a new technical result consists in the stimulation of wound healing, for the specialist is not obvious.

The new properties are not derived explicitly from the prior art in this field and is not found in the patent and scientific literature. The present invention can be used in medicine.

Based on the above, you should consider the claimed solution meets the criteria of "Novelty", "Inventive step", "Industrial applicability".

The experiments were conducted on 86 outbred mice-males. Animals obtained from the nursery of the Department of experimental biomedical modeling NII pharmacology" SB RAMS.

Studies were performed in accordance with good laboratory practice (GLP), the Order of healthcare of the Russian Federation No. 708n from 23.08.2010 "On approval of rules of good laboratory practice", the Federal Law of April 12, 2010 №61-FZ "On circulation of medicines", "Manual on experimental (preclinical) study of new pharmacological substances" (Moscow, 2005).

Example 1

Wound healing activity of the proposed tools and the mechanisms of its action was studied on the model of plane skin wounds [11]. On dailywinner the plot spins in mice under light ether anaesthesia cut the gloss is t leather size 10×10 mm To simulate a longer healing scab with experimental wounds regularly (through the day) was removed.

The proposed tool was obtained from the herb Aconite baikalskogo and iokasti high. The aboveground part of the plants collected during the flowering period in the Irkutsk region, were ground to a particle size less than 5 mm, were treated with sodium carbonate solution and was subjected to continuous extraction with chloroform for 5 days. The chloroform extract was evaporated to small volume and carefully was extracted with 5% sulfuric acid. The acid extract was podslushivaet with sodium carbonate to pH 9-10 and extracted first with ether. The ether extract was evaporated to dryness, dissolved in a small amount of ether and was chromatographically on deactivated alumina in the system hexane-acetone (90→50%). Thus hypaconitine, additionally purified by recrystallization from a mixture of the same solvent, was elyuirovaniya after mesaconitine. The substance was dissolved in distilled water to a final concentration of 0,00025%.

The resulting tool was applied topically, beginning with the first day after modeling the wound daily for the entire period of healing. The solution was applied in a volume of 30 ál. As a means of comparison used gel "Solcoseryl" (ICN Switzerland AG, Switzerland) and sea Buckthorn oil" (OAO Nizhpharm, Russia), to the which used the same pattern and in an equivalent amount, as the solution hypaconitine.

Criteria stimulating regeneration actions served the average diameter of the wound and the results of histological examination of biopsy specimens of the skin of mice, received on the 15th day of the edges of the wound defect. Biopsy samples of the wound defect of the skin were stained with hematoxylin-eosin. In each preparation was evaluated: the relief regenerate the skin, the degree and nature of infiltration, the presence and severity of edema, the number of newly formed blood vessels, hair follicles and sweat glands. Studied such pathological processes as acanthosis, hyperkeratosis, diskeratoz [12]. On the 3rd and 5th day method clone was determined by the content of mesenchymal precursor cells (CFU-f) in wound surface [13], as well as the method of colony-in test system studied the production of humoral factors stromal skin cells (colony-forming activity of supernatants adgesiruta cells from the wound surface in respect of CFU-f in the bone marrow) [13]. In addition, it was determined the direct effect of hypaconitine on CFU-F. For this purpose it has made to the culture of myelokaryocytes at a concentration of 100 nmol/ml, and then on the 7th day, counted the number of colonies [13]. Analysis was performed by the method of variation statistics using t student test and non-parametric U criterion of Mann-Whitney.

In x is de experiment wound healing in control animals was observed for 18 days experience. Using hypaconitine contributed to a significant acceleration of the process of regeneration. Healing of the defect using the proposed tools were observed for 14 days experience, while when using tools such comparison, there were only 16 days. In the experimental group (hypaconitine) had the most pronounced reduction of RAS (table 1).

Histological examination of the processes occurring in the field of wound defect in the skin of mice, showed that in the control group was the formation of defective regenerate. In most cases, the surface epithelium was observed not all over, met small areas of detachment of the epidermis from the dermis to the presence of subepidermal clefts, there was a sharp thickening of the epithelial layer with fuzzy differentiation of layers and the increase in the number of basal, granular and Horny layers. Vnutriepetelialnaya showed neutrophilic leukocytes with the formation of microabscesses were observed to have mild effects acanthosis, Hyper - and dyskeratosis. Be the fabric was presented maturing granulations with moderate histiocytoma-leukocytic infiltration, the phenomenon of swelling and foci of hemorrhage in the upper layers of the dermis. All samples in the contiguous zone of the tissue defect, there is little if estvo hair follicles and sweat glands. Reclaim skin in animals of the control group was characterized by moderate inflammation, which led to enhanced proliferation of the epithelium, which is characterized by atypical proliferation and epithelial changes in the form of acanthosis, Hyper - and dyskeratosis. The study of histostructure wound defect in the group of animals treated with therapy hypaconitine, showed the formation of full-fledged connective tissue scar, covered with epithelium. In the remaining samples (comparison group) the epithelial layer was sharply uneven, with thickening in the area of the defect at the expense of the basal layer cells. Subject fabric - granulation, sometimes with focal moderate neutrophilic infiltration. In the area adjacent to the defect in all tissue samples were observed moderate amount of hair follicles and sweat glands. The number of animals which have complete epithelialization using the proposed tool was significantly higher than in control (table 2).

Investigation of the mechanisms of regenerative activity of this tool revealed a number of unique phenomena. The use of the alkaloid was accompanied by a significant increase in the number of CFU-f in the wound surface (table 3) and accelerate their differentiation (table 4). When the identified changes were observed on the background of increasing production of growth factors, stromal to Adami skin (table 5). At the same time was noted and the direct action of this alkaloid on progenitor cells. His introduction to the culture of myelokaryocytes was accompanied by a significant increase in methylcellulose medium number of CFU-f (table 6). This comparison tool on these parameters influence did not (tables 3-6).

In General, the results demonstrate the ability of hypaconitine significantly stimulate the healing process. Mechanisms of action of the alkaloid is the activation of resident mesenchymal precursor cells, associated with its direct effect on progenitor cells, and is mediated by increased production of growth factors, stromal cells of the skin effect.

Table 1
Dynamics of the average diameter of the Russian Academy of Sciences, in cm (X±t)
Study periods/dayControlHypaconitineSolcoserylSea buckthorn oil
11,09±0,011,12±0,021,06±0,031,03±0,02
3 0,98±0,020,94±0,031,0±0,020,92±0,04
50,9±0,020,79±0,02*0,78±0,03*0,9±0,03
70,76±0,030,61±0,040,73±0,020,67±0,02*
90,57±0,020,52±0,030,51±0,01*0,54±0,03
120,28±0,030,17±0,02*0,21±0,01*0,2±0,01*
130,20±0,020,06±0,01*0,14±0,01*0,16±0,02
140,1±0,030,0±0,00,07±0,010,06±0,02
160,06±0,030,0±0,00,0±0,00,0±0,0
* checked the accuracy relative to the control at p<0,05

Table 2
The proportion of animals with complete epithelialization places of the skin defect, in % (X±m)
ControlHypaconitineSolcoserylSea buckthorn oil
4,6±0,9and 91.2±5,47*of 56.4±10,3*51,7±9,4*
* checked the accuracy relative to the control at p<0,05

Table 3
The content of mesenchymal precursor cells in the wound surface, 2.5×105the nuclears, (X±m)
Study periods/dayControlHypaconitineSolcoserylSea buckthorn oil
30,67±0,212,83±0,31*0,7±0,3 0,65±0,19
51,17±0,33,0±0,37*1,0±0,220,92±0,43
* checked the accuracy relative to the control at p<0,05

Table 4
The index of differentiation of progenitor cells (ratio klasteroobrazuyuschie units of fibroblasts to colony-forming unit fibroblasts in wound surface, in the services. units, (X±m)
Study periods/dayControlHypaconitineSolcoserylSea buckthorn oil
31,33±0,211,82±0,21,34±0,191,42±0,37
51,42±0,272,4±0,26*1,36±0,221,41±0,16
* checked the accuracy relative to the control at p<0,05

Table 5
The colony-stimulating activity supernatants stromal cells of the skin, in the services. Ed. ×105, (X±m)
Study periods/dayControlHypaconitineSolcoserylSea buckthorn oil
38,08±0,5713,25±0,88*11,3±2,427,65±0,78
50,17±0,112,75±0,28*1,0±0,570,19±0,03
* checked the accuracy relative to the control at p<0,05

Table 6
The number of CFU-f in the culture of bone marrow cells adding stimulants regeneration, 2.5×105the myelokaryocytes, (X±m)
ControlHypaconitineSolcoserylSea buckthorn oil
12,25±0,8517,6±0,33*13,7±0,6410,32±0,79
* checked the accuracy relative to the control at p<0,05

Literature

1. Mashkovsky PPM Medicines: 15-ed. - M.: OO "Publishing house New Wave", 2008. - 1206 S.

2. Kotelnikov VP Wounds and their treatment. - M.: Knowledge. 1991. - 123 S.

3. Pogodaev N.N., Gapova C., Vereshchagin A.L., Gorshkov A.G., Semenov A.A. / study of the alkaloid composition of some species of Siberian aconito // Rast. resources, issue 2, 2000, s-84.

4. Babaev, A.G., Regeneration: facts and perspectives. - M.: Publishing house of the Russian Academy of medical Sciences, 2009. - 336 S.

5. Digi A.M., Sushkov G.N., Zhdanov CENTURIES and other Immobilizovannyi granulocyte colony-stimulating factor. Pharmacological properties and prospects of use. - Tomsk: LLC "Printing manufactory, 2011. - 149 C.

6. Digi A.M., Sushkov GN. Cell therapy: new approaches // Science in Russia. - M.: Nauka, 2009. Tom. 169. No. 1. - P.4-8.

7. Nesterov Y., Povetieva T.N., Nagorniak YG, Perov A.V., Andreev TI, Roslyakova H.E., Suslov N. Mechanisms of the influence of the complex and the selected substances on reparative activity of tissues in the experiment // Experimental and clinical pharmacology. - 2009. - C, No. 3. - P.40-43.

8. Muravyova D.A., samylina I.A. Yakovlev G.P. Pharmacognosy: a Textbook 3rd ed. M: IU is Irina, 2002.

9. Osadchy S.A., Ganbaatar J., Schulz EE, Tolstikov G.A. Alkaloids Siberian species of siwakoti and Aconite and their transformation // proceedings of the First International conference "Chemistry and biological activity of nitrogen heterocycles and alkaloids" (volume 1) Moscow, October 9-12, 2001

10. Alferov A.N. A fighter for life. Aconite / Anglefire. - SPb.: ID "Whole", 2002. - 192 S.

11. Manual on experimental (preclinical) study of new pharmacological substances / Under the General editorship of the corresponding member. Russian Academy of medical Sciences, Professor Rugarama. - 2-ed., Rev. and supplementary): JSC "Publishing house "Medicine", 2005. - 832 S.

12. Microscopic techniques / edited Dscherbinova. M., 1996.

13. Goldberg ED, Digi A.M., Shah V.P. Methods of tissue culture in Hematology. Tomsk, 1992.

The use of hypaconitine as a means of having wound healing activity.



 

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