RU2620564C2 - Means of antimicrobial, reparative and anti-inflammatory action - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: means includes a dry extract from shoots of golden rhododendron containing a sum of simple phenols and phenolic glycosides of at least 28%, obtained by raw materials extraction with a particle size of 2-3 mm with water purified at raw material and extractant ratio of 1:10, extraction time for the first phase contact is 2 hours, for the second - 1.5 hours, for the third - 1 hour, with raw material stirring and temperature of 60±5°C.

EFFECT: invention has a pronounced antimicrobial, reparative and wound healing activity.

2 cl, 1 dwg, 11 tbl, 1 ex

Description

Description of the invention

The invention relates to the pharmaceutical industry, in particular to the creation of agents with antimicrobial, reparative and wound healing effects. The product contains the following ingredients: dry golden rhododendron extract, T-2 emulsifier, petroleum jelly, purified water. The tool expands the range of wound healing products of plant origin, has antimicrobial, reparative and wound healing effects.

The invention relates to the field of medicine, pharmacy, pharmacognosy, to drugs with a pronounced wound healing effect.

In the treatment of purulent-inflammatory diseases and sepsis, the fight against pathogens is of great importance. It is generally accepted that a clean, linear wound should be protected from infection, since healing of an infected wound is slower.

The decrease in the effectiveness of antibiotic therapy, the intensive increase in the number of postoperative complications of wounds, the development of allergic reactions to drugs containing antibiotics, necessitate the creation of new effective drugs for the treatment of infectious and inflammatory skin diseases and wound healing. One of the ways to solve this problem is to use herbal preparations, which are low toxic, do not cause allergic reactions and have small side effects.

Known "Wound healing agent", which is an ointment containing an extract of dry sphagnum moss, dimexide and polyethylene glycol [RF patent 2396972, A61K 36/902. Authors: Dmitruk S.E., Babeshina L.G., Dmitruk B.C.].

It is known "An agent with anti-inflammatory, antiseptic and regenerative effects", which is an emulsion containing alcoholic extracts of chamomile and calendula, aluminum hydroxide, xeroform, methyluracil. The specified tool is applied in a thin layer to the affected areas 1-2 times within 7-10 days. In this case, no side effects were detected, the emulsion accelerates healing of wounds of various etiologies [RF patent 2159113, A61K A61P. Authors: Gurbanova Sh.I., Gurbanov VV].

It is known "A drug with a regenerating and wound healing effect" containing the enzyme preparation pancreatin, methyluracil and polyethylene oxide. This ointment actively affects the wound microflora, stimulates the growth and maturation of granulation tissue and increases the epithelization rate [RF patent 2191574, A61K 31/00, A61P 17/02. Authors: Polunina E.E., Novoselova EA, Yafarov I.Sh.].

It is known "An agent with anti-inflammatory and regenerative effects." The product contains an extract of coniferous herbs, peeled cedar resin (pine, spruce, fir, larch), mummy, propolis, fir essential oil and vegetable oil. This tool is recommended for external use in medicine and veterinary medicine [RF patent 2418600, A61K 36/15, A61K 35/64, A61K 37/08. Authors: Shilo O.Yu., Kokhanova EV].

The closest is a "therapeutic and prophylactic agent with wound healing and antimicrobial effects" [RF patent 2153349, A61K 35/78. Authors: Bashura A.G., Glushko S.N.]. The product consists of a liquid (thick or dry) extract from the leaves of a walnut, obtained using 35-45% ethyl alcohol. The specified tool exhibits pronounced antimicrobial activity to a wide range of gram-positive and gram-negative test microorganisms {Bacillus subtilis, Pseudomonas aeruginosa, fungi of the genus Candida).

The purpose of the invention is the development of herbal remedies with antimicrobial, reparative and wound healing effects.

The technical result of the invention is the expansion of the assortment of herbal medicines with antimicrobial, reparative and wound healing effects through the use of dry extract from golden shoots of rhododendron; an increase in the pharmacological activity of the agent is achieved by increasing the yield of biologically active substances from raw materials.

The technical result is achieved by the fact that the shoots of golden rhododendron are extracted with purified water by the method of fractional maceration in three stages under dynamic conditions; extract drying is carried out in a spray dryer; an ointment is prepared containing the following ingredients: dry golden rhododendron extract (ESRZ) - 10.0 g, petroleum jelly - 54.0 g, purified water - 27.0 g, T-2 emulsifier - 9.0 g.

Golden Rhododendron {Rhododendron aureum Georgi.) Of the heather family (Ericaceae), is a shrub up to 50 cm tall with thick leathery leaves. It grows in the dark coniferous forests of the forest belt and on the moist rocky slopes of the high mountain belt in the mountains of the East Sayan, in Altai. There are sufficient raw materials for this plant. Elevated organs contain tannins 17%, simple phenolic compounds - rhododendrin, rhododendrol, 5-methoxy-1,3-hydroxybenzene, flavonoids - quercetin, hyperazide [4, 5, 6, 8].

The extraction conditions for shoots of golden rhododendron are selected experimentally and are shown in tables 1-7.

New is that purified water is used as an extractant, the degree of grinding of raw materials is 2.0-3.0 mm, the ratio of raw materials to extractant is 1:10 at all three stages of extraction.

New is the increase in the efficiency of the extraction process as a result of the introduction of the temperature factor (60 ± 5 ° C) and hydrodynamic conditions.

New is that the experimentally established duration of the stages of extraction - 2 hours, 1.5 hours, 1 hour provides a reduction in labor costs (in the known method - 24 hours, 6 hours, 6 hours). At the same time, the efficiency of the proposed extraction method is: for extractive substances - 84.7%, for the sum of phenols and phenological glycosides - 98.768, for the sum of flavonoids - 96.20% (Table 8).

New is the introduction of 10% in the ointment base ESRZ.

Upon receipt of the dry extract from the shoots of golden rhododendron by the known method when extraction with 45% ethyl alcohol, the efficiency of the extraction process for extractive substances was only 80.7%, for the sum of phenolic compounds - 86.4%, for the sum of flavonoids - 96.40% (table . 8).

The quantitative content of the sum of phenols and phenological glycosides was determined spectrophotometrically.

About 0.3 g (accurately weighed) of the dry extract is placed in a volumetric flask with a capacity of 200 ml, 150 ml of purified water are added and heated in a water bath until completely dissolved, cooled and adjusted to the mark with water. Take 25 ml from the flask and transfer to a 100 ml volumetric flask. To the resulting solution was added 1 ml of an 8% solution of lead acetate of basic and heated on a tile, maintaining a weak boil for 10 minutes (until complete formation of a precipitate). Then add 5 ml of 10% sodium sulfate solution (to remove excess lead ions). Next, the flask was cooled to room temperature for 1 hour and the volume of the solution was adjusted to the mark, stirred and filtered through a double paper filter into a dry flask. The first 10-15 ml of the filtrate is discarded.

The optical density of the resulting solution is measured on a spectrophotometer at a wavelength of 276 nm in a cuvette with a layer thickness of 1 cm. Purified water is used as a comparison solution. The content of the sum of phenols and phenological glycosides in terms of rhododendrin, in percent, is calculated by the formula:

,

,

Where

D is the optical density of the test solution;

47.2 is the specific absorption rate of rhododendrin (E ^1% _1cm );

m is the mass of dry extract, in g;

W is the loss in mass upon drying of the extract, in%.

The total flavonoid content was determined by differential UV spectrophotometry according to GP XI [1, 2]. In ESRZ contains the sum of phenols and phenological glycosides 25.35-29.22%; the amount of flavonoids 1.47-1.62%; the moisture content of 2.92-4.61%. In ESRH should be the sum of phenols and phenological glycosides of not less than 28% (table. 7).

What is new is the use of an ointment with a content of 10% dry golden rhododendron extract as a dosage form.

Example. Description of the technological process for obtaining dry extract:

1st stage. 1 kg of golden rhododendron shoots with a particle size of 2-3 mm are placed in the extractor, 10 l of purified water is poured. Heated to 60 ± 5 ° C and stirred for the entire extraction time - 2 hours. The extraction is filtered on a filter, measure the volume of the drained extraction (V ₁ = 6.7 l). The meal from the filter is returned to the extractor.

2 stage. In the extractor with meal remaining after the first extraction, it is poured in a volume equal to the volume of the drained extraction in the first stage (6.7 L). The extraction is carried out for 1.5 hours under the above conditions. Filter, measure the volume of the drained extract (V ₂ = 6.5 l). The meal from the filter is returned to the extractor.

3 stage. The purified water in a volume of 6.5 l is poured into the extractor with meal remaining after the second extraction. Extraction is carried out for 1 h under the same conditions. Filter, measure the volume of the drained extract (V ₃ = 6.3 L).

4 stage. The extracts V ₁ + V ₂ + V ₃ = 6.7 L + 6.5 L + 6.3 L are combined and 19.5 L extract is obtained.

Next, the extraction is defended at a temperature of + 10 ° C for 3 days, filtered through a filter-filter. The filtrate is fed into a vacuum spray dryer to a residual moisture content of dry extract of not more than 5.0%.

5 stage. Spent meal is thrown away.

Description of the technological process of obtaining ointments [7]:

1. In a container 1 (evaporating cup), a T-2 emulsifier 9.0 g, Vaseline 54.0 g (60 ° C) are melted (80 ° С) in a water bath, quickly transferred to a micronizer and mixed for 2 minutes.

2. Dissolve 10.0 g of ESRS in 27 ml of purified hot water (60 ° C) in a container 2 (stand).

3. The resulting aqueous solution of dry extract is introduced into the molten base and homogenized on a tissue micro-grinder for 3 minutes.

The quantitative content of the sum of phenols and phenological glycosides in the ointment was determined by spectrophotometric method.

About 1.0 g (accurately weighed) of the ointment is placed in a 200 ml flask, 20 ml of purified water is added, heated in a water bath, cooled under a stream of cold water (without shaking), filtered through a paper filter into a 100 ml volumetric flask. The operation is repeated 3 times. To the resulting solution was added 1 ml of an 8% solution of basic acetate of lead and heated on a tile, maintaining a weak boil for 10 minutes (until complete formation of a precipitate). Then add 5 ml of a 10% sodium sulfate solution (to remove excess lead ions). Next, the flask was cooled to room temperature for 1 hour and the volume of the solution was adjusted to the mark, stirred and filtered through a double paper filter into a dry flask. The first 10-15 ml of the filtrate is discarded.

The optical density of the resulting solution is measured on a spectrophotometer at a wavelength of 276 nm in a cuvette with a layer thickness of 1 cm. A comparison solution is prepared according to the procedure described above from a sample of ointment prepared without dry extract.

The content of the sum of phenols and phenological glycosides in terms of rhododendrin in ointments with REE in percent (X) is calculated by the formula:

;

;

Where

D is the optical density of the test solution;

47.2 is the specific absorption rate of rhododendrin (E ^1% _1cm );

m is the mass of ointment, in

The quantitative content of the sum of phenols and phenological glycosides in the ointment is 2.52-2.65%.

The antimicrobial activity of the dry extract and ointment was studied on 12 strains of gram-positive and gram-negative microorganisms in a dense nutrient medium using the "hole" method. The activity of the drugs was judged by the diameter of the sterile zone, taking the following notation: 10 mm - “+”, 10-15 mm - “++”, 15-20 mm - “+++”, 25-30 mm - “++ ++ ". Dry extract and ointment exhibit antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Pseudomonas aeruginosa, Salmonella typhymurium (Table 9).

The wound healing effect was studied on the model of an infected wound caused by the introduction of a laboratory strain of Staphylococcus aureus. The experiments were carried out on 30 white rats weighing 200-230 g, both sexes of the Wistar line contained in standard vivarium conditions. The animals were kept under standard conditions in accordance with the rules approved by the Ministry of Health of the USSR on 07/06/1973, and in accordance with the EU Council Directive of 11/24/1986 on the protection of animals used for experimental and other scientific purposes. Animals received a standard diet (PK-120-1).

The study of the wound healing effect of the ointment with ESRD was carried out in comparison with Calendula ointment 10% (registration number 79/301/5), which is used in the treatment of burns, non-healing wounds and fistulas. Ointment "Calendula" has anti-inflammatory, antimicrobial and reparative effects [3].

After thiopental sodium anesthesia and skin preparation, a linear wound was applied to rats on the back along the spine to a muscle layer of 3 cm in length. Then 0.2 ml of suspension was applied to the wounds, which was passivated for a long time on nutrient media of a laboratory strain of Staphylococcus aureus containing 1 ml 10 ⁹ microbial bodies (concentration was determined according to the turbidity standard). After this, the wounds were sutured with adaptive sutures with silk No. 2.

The animals were divided into 3 groups of 10 rats each:

Group 1 (control) - rats with a full-layer skin wound infected with strains of St.aureus, the wound healed naturally;

Group 2 (experimental) - rats with a full-layer skin wound infected with strains of St. aureus treated with ointment with ESRD 10%;

Group 3 (comparisons) - rats with a full-layer skin wound infected with St. aureus treated with Calendula Ointment 10%.

The presence of local purulent process was evaluated on 3, 7 and 14 days. The control time points were determined by the fact that the wound acquires signs of acute inflammation after 72 hours.

Particular attention was paid to signs of inflammation from the side of the wound - hyperemia, pastiness of the edges, condition of the sutures, separated from the wound (mainly purulent).

Animals were withdrawn from the experiment on the 14th day after intraperitoneal administration of a lethal dose of thiopental sodium.

For early tensiometric and histological examination in the transverse direction, strips of tissue 0.5 cm wide were taken from the excised wounds. The biomechanical properties of the healing wound were studied by determining the strength of the adhesion of its edges in grams. For this purpose, we used a universal device for mechanical and thermomechanical testing of polymer fibers and films of the UMIV-3 model (Russia).

For histological examination of tissue from wounds, sections of preparations fixed in formalin were stained with hematoxylin and picrofuxin.

Statistical processing of the obtained data was carried out using the Statistica 6.0 program, designed to evaluate the results of medical and biological observations, with calculation of the arithmetic mean (M), standard deviation (σ), arithmetic mean error (m), and confidence coefficient (t ), an indicator of probability (p).

Statistical data processing was performed using a standard MS Excel data processing program.

The research results indicate that on the 3rd day of the experiment, rats in the control group showed hyperemia and pasty of the edges of the wound, profuse purulent discharge with an unpleasant odor (about 3 ml), signs of intoxication (adynamia, thirst, poor appetite). The bottom of the wound is moist, hyperemic, with overlays of fibrin. In rats of the experimental group and the comparison group, these phenomena were observed in 80% of animals - mild hyperemia and swelling of the edges of the wound, a small amount of purulent discharge without a characteristic odor, weak signs of intoxication. Moreover, the size of the wounds in rats of the control, experimental and comparison groups 2.5 cm long, 0.1 cm wide, with sutures.

On the 7th day of the experiment, the animals of the control group and the comparison group retained clinical signs of purulent inflammation, however, edema and the amount of purulent discharge from under the formed scab decreased. In rats of the experimental group there was a slight weeping of odorless wounds. Microbiological studies have shown the absence of Staphylococcus aureus in the wound discharge. The size of the wounds in rats of the 1st group is 2.5 cm long, 0.4 cm wide (diastasis of the wound edges), in rats of the 2nd and 3rd groups - 2.5 cm, 0.1 cm wide (there is no diastasis of the edges). Moreover, in all groups, the bottom of the wound was cleaned and represented by granulation tissue.

On the 14th day of the experiment, in rats of the 2nd and 3rd groups, the wounds were completely epithelized, while in the 1st group 10% of animals remained with less pronounced epithelization and partial scab conservation. In general, the healing of a wound defect in all groups of animals in the form of a scar.

The results of the studies indicate that the ointment with SERH increases the strength of the postoperative scar by 4.1 times on the 14th day in comparison with the control group. At the same time, the comparison drug Calendula ointment increased the strength of the postoperative scar on the 14th day by 3.6 times in comparison with the control group (Table 10). The ointment with ESRP exceeds Calendula ointment by 12.9% in terms of the strength of the resulting postoperative scar (Fig. 1).

Thus, as a result of the studies, it was found that the wound healing effect of the ointment with SERH is not inferior to the comparison drug - ointment with calendula extract.

1. State Pharmacopoeia of the USSR: issue. 1. General methods of analysis / Ministry of Health of the USSR. - 11th ed., Ext. - M .: Medicine, 1987 .-- 336 p.

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5. Mirovich V.M. Resource characteristics of rhododendron species in the territory of the Irkutsk region and the western part of the Republic of Buryatia / V.M. Mirovich, G.M. Fedoseeva G.I. Bocharova // Development, research and marketing of new pharmaceutical products: Sat. scientific works. Vol. 64. - Pyatigorsk, 2009 .-- S. 85-87.

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Claims (6)

1. An agent that has an antimicrobial, reparative and wound healing effect, based on a plant extract, characterized in that it contains dry extract from shoots of golden rhododendron with a total content of at least 28% of phenols and phenol glycosides obtained by extraction of raw materials with a particle size of 2- 3 mm of purified water with a ratio of raw materials and extractant 1:10, the extraction time for the first contact of the phases is 2 hours, the second is 1.5 hours, the third is 1 hour, with stirring of the raw materials and a temperature of 60 ± 5 ° C. 2. The tool according to claim 1, characterized in that it contains an extract of dry golden rhododendron, petroleum jelly, emulsifier T-2 in the following ratios, wt. %: dry golden rhododendron extract 10, petroleum jelly 54, emulsifier T-2 9, purified water 27.

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