RU2714930C2 - Agent exhibiting wound healing activity - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: invention refers to pharmaceutical industry, namely to application of dry extract of medicinal leaves and flowers as a wound healing agent. Application of dry extract of leaves and flowers of drug hedge hyssop as a wound healing agent obtained by way of crushing leaves and flowers of medicinal drug, extraction thereof with 96 % alcohol in a water bath until boiling and boiling for 14–15 minutes, evaporation at temperature 55–60 °C, diluting the evaporated residue first with distilled water at temperature of 40–50 °C, then adding chloroform in ratio of 4/5 part of water and 1/5 part of chloroform, cooling to room temperature and centrifugation at 1500 rpm for 15 minutes, followed by separation of the aqueous fraction and drying it.

EFFECT: above described application of dry extract of leaves and flowers of drug hedge hyssop enables to significantly reduce the length of healing of infected wounds.

1 cl, 2 dwg, 3 ex

Description

The invention relates to the pharmaceutical industry and regenerative medicine, namely to the creation of medicines based on Novogalennogo drug, to stimulate the healing process. The invention can significantly reduce the healing time of infected wounds.

A wound (vulnus) is called any mechanical damage to the body, accompanied by a violation of the integrity of the integumentary tissues - the skin or mucous membranes.

Surgical wounds are inflicted intentionally, for medical or diagnostic purposes, in special aseptic conditions, with minimal tissue trauma, with pain relief, with thorough hemostasis and, as a rule, with matching and joining of dissected anatomical structures with sutures. Thus, there is no pain with surgical wounds, the possibility of bleeding is minimized, and the gaping is usually eliminated at the end of the operation by suturing, i.e. the main signs of a wound are artificially eliminated. Due to its characteristics, surgical wounds are prone to primary healing. <...> Regardless of the nature and location, all random wounds are bacterially contaminated. However, in addition to the initial contact of microorganisms in the wound, their further penetration from the patient’s skin, from the surrounding air, from various objects is possible. (General surgery: textbook / Petrov S.V. - 3rd ed., Rev. And add. - 2010. - 768 p.: Ill.)

Bacterial contaminated wounds are characterized by the presence of various microorganisms. Primary microbial contamination occurs at the time of injury. Secondary microbial contamination occurs during treatment. Wound microflora in these cases are called microorganisms that grow in the wound, but do not cause pathogenic effects.

<...> The complexity, multifactorial nature of the etiology and pathogenesis of wound infection requires the use of a system of preventive and therapeutic measures aimed at: cleaning the wound from necrotic and non-viable tissues; restoration of viability of tissues surrounding the necrosis zone by eliminating edema, restoration of microcirculation, normalization of biochemical processes; elimination of systemic disorders (microcirculation, proteolysis, metabolic processes, pulmonary, granulomonopoiesis, immunogenesis); suppression of pathogens of wound infection. (Bordakov, V.N. B82, Wound. Wound healing process. Principles of wound treatment: textbook.-method. Allowance / V.N. Bordakov. - Minsk: BSMU, 2014. - 31 p.).

Thus, the treatment of the wound process is a complex task, solved not only by surgery, but also requiring adequate conservative support. Moreover, drugs for the treatment of wounds should have anti-inflammatory, antimicrobial and wound healing properties.

Known antibacterial and regenerating agent, made in the form of an oily gel for external use "Fusidin" (RU Patent No. 2512757) with anti-inflammatory, antibacterial and wound healing effects.

However, the known drug has the following disadvantages: firstly, it reduces the body's immune reactivity; secondly, synthetic and expensive components are required to produce it.

The plantain drug is also known (Mashkovsky M. D. Medicines. M. Medicine, 1993), the active substance of which is plantaglucid (RU 185456), which refers to practically non-toxic drugs (Sosnina S. A. Comparative pharmacognostic study, standardization of raw materials and phytopreparations Species of the genus plantago 1. ”Abstract, Perm 2009). However, the wound healing activity of this composition is low. The analogue can only be used as an anti-inflammatory agent.

The Abisil-1 agent (RU 2054945) possesses the indicated properties, the disadvantages of this drug include the low ability to stimulate the growth of granulation tissue, as well as the impossibility of accurate dosing of the active substance and, as a consequence, the unpredictability of maximum pharmacological activity.

Thus, at present, all existing means have a number of disadvantages and are characterized by low ability to wound healing.

Medicinal Avran (Gratiola officinalis L.), collected in an ecologically clean area, on the island of Chardym in the Saratov region. The extract was obtained by us by the author's method (RF Patent 2482863), which allows to significantly increase the yield of bioflavonoids and provides a minimum yield of toxic compounds (alkaloids, glycosides, etc.), which is especially important when obtaining non-toxic extracts of poisonous plants, which include medicinal avran. Previously, betulinic acid with antitumor activity was described in the composition of avrana grass, but the technology we used to obtain the extract excluded the yield of this compound. The extract obtained by this method from Avrana officinalis has the following chemical composition: 4-vinyl-2-methoxyphenol; 2,3-dihydro-3,5-dihydroxy-6-methyl-4H-pyran-4-one; 2,3-dihydrobenzofuran; 3-furancarboxylic acid; 5-hydroxymethyl-2-furaldehyde; ethyl a-d-riboside; 4-propylphenol; catechol; L-Luxose (pentose); 6-deoxyhexose L-galactose; benzoyl acetic acid ethyl ester; hexadecanoic acid (palmitic acid); homovanilinic acid; glucose; 1,4-anhydro-d-mannitol; benzoic acid; quercetin. The average value of quercetin in this extract using the calibration graph of a standard sample of quercetin (98%) Sigma is 0.66%. The amount of quercetin in the dry residue of extractives (obtained from 10 g of dry grass of Avran) that we established by liquid chromatography was 350 μg. The extract was obtained at the Department of General Biology, Pharmacognosy and Botany, S.I. Razumovsky SSMU, was administered to animals in the form of an aqueous solution (extract concentration of 100 mg / ml). In experiments on rats, the effect of the aqueous extract of avrana medicinal on the level of dioxidine-induced lipid peroxidation in the blood was established. The ability of avran extract to reduce the content of molecules of average weight by 18% (MSM) and malondialdehyde by 49% (MDA) was revealed only with intramuscular injection.

These data indicate a decrease in peroxidation under the influence of an external factor (Navolokin N.A., Polukonova N.V., Maslyakova G.N., Bucharskaya A.B., Durnova N.A. Saratov Journal of Medical Scientific Research. 2013. T . 9, No. 2. S. 213-220).

Previously, we established anti-inflammatory and antimicrobial activity of Avran with intramuscular administration (Anti-inflammatory, antipyretic and antimicrobial activity of flavanoid-containing extract of medicinal Avran (gratiola officinalis 1.), Polukonova N.V., Navolokin N.A., Raikova S.V., Maslyakova G. .N., Bucharskaya A.B., Durnova N.A., Shub G.M., Experimental and Clinical Pharmacology vol. 78, p. 34-38, 2015).

The technical task of the present invention is to expand the arsenal of domestic products based on natural plant-based raw materials of complex action with wound healing activity.

The advantages of the proposed new wound healing drug, created on the basis of avran extract, are low toxicity, the availability of raw materials from which the product is obtained, as well as a wide range of positive effects.

So, the drug has its own anti-inflammatory effect and the ability to reduce the level of endogenous intoxication. The pathogenetic mechanism of the therapeutic effect is a decrease in the severity of local inflammation and a decrease in endogenous intoxication, which is confirmed by a decrease in the levels of malondialdehyde (MDA), lipid hydroperoxides (GPL), and medium-weight molecules (MSM) that accumulate in the blood when tissue cells die. The work demonstrated the possibilities of both oral, intramuscular and local methods of using the drug.

We first established a new wound healing property of the drug. The new wound healing drug is an evaporated extract of the leaves of the drug Avran obtained by grinding them, extraction with alcohol 96%, evaporation of the extract, adding chloroform, centrifugation and separation into aqueous and chloroform fractions, followed by removal of the chloroform fraction. Extraction with alcohol is carried out in a water bath to a boil and boiled for 14-15 minutes, evaporated at a temperature of 55-60 ° C. The evaporated extract is first diluted with distilled water at 40-50 ° C, then chloroform is added in a proportion of 4/5 parts of water and 1/5 part of chloroform, cooled to room temperature and centrifuged at a speed of 1500 rpm for 15 minutes, then the aqueous fraction is separated and dried it (RU 2482863).

The product is an evaporated viscous transparent extract with a pleasant tart pungent odor, light yellow in color, mixes with water and ethyl alcohol in any ratio.

The extract thus obtained is a biologically active composition with new pharmacological properties, namely, along with anti-inflammatory, antimicrobial and previously unknown for both the Avrana medicinal plant and the wound healing effect described above.

Authenticity and quality parameters of the product. Collection of material. The material was the raw material of Avrana officinalis, collected in the Voskresensky district of the Saratov region in the vicinity of s. Chardym in July 2016. Dried leaves of medicinal avran were pre-crushed for a more complete extraction.

Obtained by the patented method (RF Patent 2482863), one stripped off extract of flowers and leaves of avran is non-toxic, which is due to the chemical composition and has been previously confirmed in rats [Navolokin N.A., Polukonova N.V., 2013, 2014].

We first conducted experimental work to establish a wound healing effect.

In an experiment conducted in accordance with the guidelines for the experimental (preclinical) study of new pharmacological substances [Guidelines for preclinical studies of drugs. Part one. / Ed. A.N. Mironova. - M.: GrifiK, 2012. - 944 p.], 40 male white laboratory rats weighing 150 ± 50 g were used, which under the condition of anesthesia in the interscapular region under non-sterile conditions were inflicted the same wounds, according to a special stencil. Animals with experimental non-sterile wounds were randomly divided into four groups of 10 rats - the first, second and third experimental, receiving topical, intramuscular and oral abrana extract in a dosage of 1 g / kg and the fourth - comparison group, animals with non-sterile wound, daily treated with a 3% hydrogen peroxide solution. In the experimental groups, the solution was administered to rats locally (in the first group), intramuscularly (in the second group) and orally (in the third group), daily, until the wounds had completely healed.

The dynamics of wound healing was evaluated by changing its area according to the formula: S = A * B * π / 4 (mm ² ), where A is the width, B is the length of the wound in mm (Median of the size of the wound in all groups was 374 mm ² ). Measurements were carried out with an electronic caliper once every two days from the start of the experiment. The proportion of wound healing was calculated using the formula C = (S _beg -S ₁ / S _beg ) * 100%, where S _beg is the initial area of the wound, S ₁ is the area of the wound on the current day of measurement.

Changes in the dynamics of wound healing.

The Figure 1 shows the dynamics of changes in the morphology of wound defects. An assessment of changes in the morphology of wounds on the 3rd day of the experiment revealed that no microbial complications were observed in the wounds of animals of all groups of microbial complications. The average area of the wounds of animals of the comparison group was 379.68 ± 26.5 mm ² , in the group with local exposure to Avrana extract 354.55 ± 31.5 mm ² , with intramuscular injection - 339.7 ± 42 mm ² , with oral administration - 352.5 ± 43 mm ² .

On the 7th day from the start of the experiment, there was no hyperemia and edema in the wounds of the animals of the comparison group, the wounds were covered with a dry scab and their average area was 297.9 ± 20.4. edema and suppuration of wounds were absent, the average area in the group with local exposure to Avrana extract was 286 ± 18.1 mm ² , with intramuscular injection - 268.9 ± 36 mm ² , with oral administration - 283.3 ± 28 mm ² . (Figure 1)

Starting from day I, there was a significant acceleration of wound healing in the group with intramuscular injection of avran extract (wound healing rate - 82.4%), compared with the comparison group (wound healing rate - 51.1%). On day 13, acceleration of wound healing was observed in the group with local exposure (the proportion of wound healing - 82.3%) compared with the comparison group (the proportion of wound healing - 51.6%). On the 15th day, acceleration of wound healing was observed in the group with local exposure (the proportion of wound healing - 82.3%) compared with the comparison group (the proportion of wound healing - 51.6%). With oral administration of the extract of Avran, a significant acceleration of healing was observed from the 15th day of the experiment (84.8%) in the experimental group versus 57.9% in the comparison group. Data on the dynamics of changes in the area of wounds in rats during treatment were systematized and reflected in Figure 2.

On the 21st day after the operation, there was a further reduction in the average area of wounds in all groups of animals: in the comparison group, it was 90.5 ± 3.4 mm ² , the average area in the group with local exposure to Avran extract 1 ± 0.5 mm ² , with intramuscular the introduction of 1.3 ± 0.4 mm ² with oral administration of 9 ± 4.2 mm ² .

When applied topically to the 19th day of the experiment, with intramuscular and oral administration of the extract, the healing was completely completed by the 21st day of the experiment.

Example 1

For the experiment was taken a white laboratory rat male, which in the interscapular region caused a non-sterile wound with an area of 361.1 mm ² . Every day until the wound healed, the animal was applied a napkin soaked in a 10% solution of Avrana officinalis extract on the wound surface.

The dynamics of wound healing was evaluated by changing its area according to the formula: S = A * B * π / 4 (mm ² ), where A is the width, B is the length of the wound in mm (Median of the size of the wound in all groups was 374 mm ² ). Measurements were carried out with an electronic caliper once every two days from the start of the experiment. The proportion of wound healing was calculated using the formula C = (S _beg -S ₁ / S _beg ) * 100%, where S _beg is the initial area of the wound, S ₁ is the area of the wound on the current day of measurement.

The following data were obtained:

The dynamics of the area of the wound (mm ² ): 1 day - 361.1; 3 day - 314; 5 day - 290.4; 7 day - 270.3; 9 day - 241.8; 11 day - 113.1; 13 day - 87.9; 15 day - 39.7; 17 day - 16.2; 19 day - 0. As a result, a reduction in the healing time of the wound by 8 days was noted, compared with the comparison group.

Example 2

For the experiment, a white laboratory male rat was taken, which inflicted an unsterile wound with an area of 402.7 mm ² in the interscapular region. Daily, before wound healing, the animal was intramuscularly injected with an aqueous solution of Avrana officinalis extract at a dosage of 1 g / kg.

The dynamics of wound healing was evaluated by changing its area according to the formula: S = A * B * π / 4 (mm ² ), where A is the width, B is the length of the wound in mm (Median of the size of the wound in all groups was 374 mm ² ). Measurements were carried out with an electronic caliper once every two days from the start of the experiment. The proportion of wound healing was calculated using the formula C = (S _beg -S ₁ / S _beg ) * 100%, where S _beg is the initial area of the wound, S ₁ is the area of the wound on the current day of measurement.

The following data were obtained:

The dynamics of the area of the wound (mm ² ): 1 day - 402.7; 3 day - 345.4; 5 day - 309.3; 7 day - 251.2; 9 day - 241.8; 11 day - 153.3; 13 day - 89.9; 15 day - 37.7; 17 day - 28.3; 19 day - 9.42; 21 days - 0. As a result, a reduction in the healing time of the wound by 6 days was noted, compared with the comparison group.

Example 3

For the experiment, a white laboratory male rat was taken, which inflicted a non-sterile wound with an area of 397.2 mm ² in the interscapular region. Daily, before wound healing, the animal was orally administered with an aqueous solution of Avrana officinalis extract at a dosage of 1 g / kg.

The dynamics of wound healing was evaluated by changing its area according to the formula: S = A * B * π / 4 (mm ² ), where A is the width, B is the length of the wound in mm (Median of the size of the wound in all groups was 374 mm ² ). Measurements were carried out with an electronic caliper once every two days from the start of the experiment. The proportion of wound healing was calculated using the formula C = (S _beg -S ₁ / S _beg ) * 100%, where S _beg is the initial area of the wound, S ₁ is the area of the wound on the current day of measurement.

The following data were obtained:

The dynamics of the area of the wound (mm ^ 2): 1 day - 413.3; 3 day - 379.9; 5 day - 343.1; 7 day - 282.6; 9 day - 241.8; 11 day - 184.7; 13 day - 101.3; 15 day - 77.7; 17 day - 57.3; 19 day - 18.84; 21 days - 0. As a result, a reduction in the healing time of the wound by 6 days was noted, compared with the comparison group.

Thus, as can be seen from the description and examples, the agent, which is a dry extract of the leaves of the drug Avran obtained by the above method, has a wound healing property. The use of the extract, regardless of the route of administration, reduced the time for wound healing by 8 to 6 days.

Claims (1)

The use of dry extract of leaves and flowers of Avrana officinalis as a wound healing agent obtained by grinding leaves and flowers of Avrana officinalis, extraction with 96% alcohol in a water bath to boiling and boiling for 14-15 minutes, evaporation at a temperature of 55-60 ° C, dilution of the evaporated residue first with distilled water at a temperature of 40-50 ° C, then adding chloroform in the proportion of 4/5 part of water and 1/5 part of chloroform, cooling to room temperature and centrifugation at a speed of 1500 rpm minute for 15 minutes, followed by separation of the aqueous fraction and drying it.

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