RU2054945C1 - Agent showing antiinflammatory, antibacterial and wound-healing activities - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: agent has components at the following ratio, wt.-%: capsule extract of Siberia fir (Abies sibiricus) 19-20; monoterpene compounds 0.02-0.20, and vegetable oil - the rest. EFFECT: enhanced antiinflammatory, antibacterial and wound-healing activities. 3 tbl

Description

 The invention relates to medicine, namely to the creation of agents with anti-inflammatory, antibacterial and wound healing activity on a plant basis.

 Among the known agents that have anti-inflammatory, antibacterial and wound healing effects, the gel "Fusidine" should be considered.

 However, the well-known drug is low active, produced from synthetic expensive products. In this case, the components included in its composition, taken separately, are toxic compounds and can help reduce the body's immune reactivity.

 Among the pharmacological agents with this activity, the closest is the preparation of plantain (Mashkovsky M. D. Medicines. M. Medicine, 1993).

 However, the activity of this drug is low.

 The aim of the invention is the creation of funds with anti-inflammatory, antibacterial and wound healing activity and containing capsular extract from Siberian fir, monoterpene compounds and vegetable oil.

 Natural capsule extract, as well as monoterpene compounds of essential oils can be obtained by known methods.

 As a third component, you can use sunflower, soybean and olive oils.

Temperature conditions 18-20 ^{° C} is optimal, because an increase in temperature causes an acceleration of the dissolution of the fraction of monoterpene compounds in the capsule extract, but increases the possible loss by evaporation of volatile components.

 Mixing the resulting substance with vegetable oils is carried out at room temperature in order to avoid evaporation of volatile fractions and inactivation of the target product.

 The choice of vegetable oils as a solvent promotes better permeability of the final product through the lipid layer of cell membranes. It is also proved that alcohol is not an ideal solvent for capsule extract (see the data of the initial physico-chemical analysis of the capsule extract in example 1).

 The search for means among the known solutions showed that the distinguishing features were not used in the known means. There were no reports of the use of a capsule extract from Siberian fir, enriched with monoterpenic compounds, the fraction of essential oil, similar capsules as a substance for the preparation when dissolved in vegetable oils. No solutions have also been identified where, as an anti-inflammatory and wound healing agent, it was proposed to use a capsular extract from Siberian fir enriched with monoterpene compounds.

 The proposed tool is obtained as follows.

Example 1. Capsule extract of Siberian fir (Abies Sibiricus Ledebur), which is a complex multicomponent mixture of biologically active essential oils, neutral diterpenes, resin acids and their esters, is obtained by direct puncture of the capsule located between the bark and the bast. The resulting extract was filtered at room temperature. It is a viscous amber-yellow liquid. Physico-chemical analysis showed:
P $\genfrac{}{}{0ex}{}{\text{20}}{\text{D}}$ 1.5112;
d $\genfrac{}{}{0ex}{}{\text{20}}{\text{n}}$ 1.0328 g / cm ³ ,
IKS 3400, 2900, 1740, 1690 1700, 1450, 1390, 1270
1250, 1040, 900 800 words cm ^-1 .

PMR: 0.82; 1.67; 4.71; 5.77 ppm (C ₆ D ₆ ).

Elemental analysis data:
C 77.82; H 10.19; S no; N no.

 The solubility is given in table. one.

 Take 1000 ml of purified capsule extract, place in a flask in a water bath with an attached Clevenger apparatus, a finger cooler and a graduated separation pipette with a drain cock. The distillation is continued for 12 hours. The volatile fraction collected in this way is an essential oil consisting of monoterpene compounds: tricyclene, limonene α-pinene, santene, camphene, etc.

1.0 ml monoterpene compounds are mixed with 195 ml of capsule extract at a temperature range of 18-20 ^{° C} with constant stirring and then dissolving the obtained substance in 804 ml sunflower oil. The obtained amber-colored product with a characteristic smell of essential oils is tested for anti-inflammatory, antibacterial and wound healing activity.

 Anti-inflammatory and wound healing activity was studied in 36 outbred rabbits weighing 2.5-3.0 kg when evaluating Abisil 1 obtained in example 1, 2, 3, in comparison with the gel "Fusidin" and other similar means.

To obtain a model of an experimental purulent wound in rabbits in the interscapular region, a skin flap with a diameter of 3-4 cm was completely excised, followed by destruction of the aponeurosis and underlying muscles with a blade. The wound was infected with an association of 2 × 10 ⁹ microbial bodies / ml Staphylococcus aureus (Gure strain) and 5 × 10 ⁹ microbial bodies / ml Klebsiella (Klebsiella pneumoniac strain 12 AC).

 The drug was used from the moment of the formation of a purulent wound (2-43 days after infection) by application to the treated wound (after removal of necrotic masses within healthy tissues) 2 times a day for 19 days, applying 1 ml of Abisil-1 (obtained in three ratios), sea buckthorn oil and 1 g of gel "Fusidine". The effectiveness of the treatment was evaluated by comparing the course of the wound process according to a complex of clinical and bacteriological data.

 Microscopic evaluation took into account the dynamics of local purulent-inflammatory phenomena (edema, hyperemia, infiltration of surrounding tissues); the degree and composition of the wound separator; purification periods; nature of granulation.

The dynamics of pathogen elimination from the wound was evaluated bacteriologically, taking into account the degree of contamination of the wound in CFU / g of tissue. The number of microbes in 1 g of tissue was calculated by the formula: H nx10x10 (or 100, or 1000 depending on the dilution x K, where
N the number of microbes per 1 g of tissue
n number of microbes grown in a cup
10 conversion per gram of suspension
10 (100 or 1000) dilution of the material sown on the cup from which the colony is counted
To the conversion factor of the sample on 1 g of biopsy.

 Studies were carried out on days 2, 4, 6, 8, 10 of treatment with drugs.

Wound healing time was determined by the formula:
(t ₁ t ₂ ) / t ₁ · 100% where t _{1 is the} time of final wound healing in the control group, days;
t ₂ time of final healing of wounds in the experimental group, days.

The area of the wound healing site was determined in percent by the formula:
100 A / S, where S is the initial area of the wound, cm ² ;
And the difference between the initial area of the wound and the area of the wound at the moment.

 Cytological examination was carried out by the method of fingerprints, which were taken from the surface of a purulent wound and at the same time as in bacteriological examination. Fat-free glass slide was applied to different parts of the wound surface.

 Microscopic examination assessed the quantitative characteristics of the cellular composition of wound exudate, counting 500 cells of different morphology with the calculation of average values.

A purulent wound before treatment in all groups had an area of about 110 cm ² with edematous infiltrated edges and hyperemia of the surrounding skin. Its bottom was covered with a bloom of fibrin with patches of necrosis and purulent discharge.

 On the 2nd day of treatment with Abisil-1, obtained according to example 1, the edema and wound infiltration in animals decreased, hyperemia decreased. In the group of animals treated with Fusidin gel, signs of inflammation were clearly expressed. At the bottom of the wound, a significant amount of fibrinous plaque, thick pus and necrotic tissue was observed. In the control group (untreated), abundant purulent discharge, bleeding, severe swelling and hyperemia of the bottom and edges of the wound were observed.

 On the 8th day in animals treated with Abisil-1, a significant cleansing of the wound from purulent-necrotic masses was observed, and by 9 days there were areas of marginal epithelization. A similar picture with the use of Fusidin gel and sea buckthorn oil is noted by 10-12 days, and in the control group (untreated) by 14 days of treatment.

 The bottom of the wound is completely filled with granulation tissue by 13-14 days, against 28 days of control.

 Studies of the anti-inflammatory and wound healing activity of the proposed drug (see tables. 2 and 3) showed that the percentage of wound healing by 17 days corresponds to 90.5 ± 5.4 versus 53.3 ± 5.1 in the control and 78.7 ± 6 , 1 using Fusidin gel, respectively.

Accordingly, the degree of contamination (CFU / g) fell. The initial degree of contamination of the wound was 10 ⁶ 10 ⁷ CFU / g. The elimination of the pathogen from the wound began with the use of all means from 4 days, while in prototypes it lasted up to 10 days and above, and with the use of Abisil-1 obtained in example 1, low titers 1 were noted already from 6 days. 8x10 ⁴ CFU / g versus 4.8x10 ⁶ in the control and 3.5x10 ⁴ using the Fusidin gel.

 In the cytological study of the course of the wound process, almost complete homogeneity of the cellular composition of animal exudate was established before treatment, which in 96.6% was determined by polymorphonuclear neutrophils, mostly degeneratively altered, with pathological granularity of the cytoplasm. A large number of microbial bodies located both intracellularly and extracellularly in a state of perverted and incomplete phagocytosis were determined in all prints.

 On the second day of treatment with Abisil-1 and the prototype, no significant differences were found in cytological studies. Cytograms in all cases are characterized as inflammatory.

 On the 12th day of treatment in the group of animals treated with Abisil-1 (ratio 19.5: 0.02: ost-e), the neutrophil content in the cytogram decreased to 55-60%, the number of macrophages increased. Microbial cells were contained in a small amount in a state of phagocytosis. In animals treated with the Fusidin gel, a smear of neutrophils contained 75-80% 5 10% of the cellular composition was accounted for by lymphocytes, monocytes and fibroblasts.

 In the control group, a large number of microorganisms are located extracellularly, and neutrophils and macrophages are in a degenerated and phagocytic form.

 On the 17th day in the imprints of the group of animals treated with Abisil-1 (corresponding to 19.5: 0.02: ost-e), young cells of granulation tissue began to predominate: profibroblasts, fibroblasts, polyblasts, macrophages. Microbial cells are absent, single neutrophils.

 A similar picture when using the gel "Fusidin" was noted by the 21st day of research, and in the control by 26 27 days.

EXAMPLE EXAMPLE 2. The method is carried out as in Example 1, with the difference that the selected fraction from the capsular extract of essential oils comprising monoterpenoids, is mixed with a capsular extract of Siberian fir at a temperature of 18-20 ^{° C} in a ratio of 0,5 ± 0 , 3 99.5 ± 0.1 (0.5%), followed by dissolution in vegetable (soybean) oil in a ratio of 1: 4 (20%).

 Already on the second day of treatment in animals when using Abisil-1, obtained according to example 2, a positive dynamics of the wound process was observed: swelling and infiltration of the wound decreased, hyperemia disappeared.

 On the 4-6 day of treatment using the funds obtained in example 2, there was a cleansing of the bottom of the wound from fibrin, necrotic tissue in the wound is practically absent. In the group (Fusidin gel), moderate pus was observed, slight swelling persisted, and necrotic masses were present in the wound.

 On day 8 in animals treated with Abisil-1, a complete cleansing of the wound from purulent-necrotic masses was observed; the wound was performed with juicy granulations with the appearance of marginal epithelialization sites. When using the Fusidin gel during these periods, the remains of necrotic masses and fibrinous plaque, which were removed mechanically during the treatment of the wound, were still preserved at the bottom of the wound.

 By 10-12 days of observation in animals using Abisil-1, the bottom of the wound was completely performed by granulation tissue with large areas of fibrosis, marginal epithelization. A similar picture in the group of animals using the gel "Fusidin" is noted at 14 to 16 days, and in the control at 17 days.

 Starting from 17-19 days in the group treated with Abisil-1, complete healing of the wound with the formation of a scar is noted. The entire wound is covered with a coat.

 An identical picture in the gel "Fusidin" is noted after 5 6 days, and in the control of 8 10.

 Studies of anti-inflammatory and wound healing activity revealed (see Tables 2 and 3) that the percentage of healing of superficial wounds by 17 days was 95.5 ± 4.4 versus 53.3 ± 5.1 in the control and 89.4 ± 5.3 using the gel "Fusidin". In the future, the term for complete wound healing using Abisil-1 ended at 18.1 ± 0.4 days, while using Fusidin gel it was 21.8 ± 0.6 versus 26.1 ± 1.5 in control.

Inoculation of microorganisms from purulent wounds by 6-8 days ended with 1.1x10 ² CFU / g using the obtained means, Fusidin gel and control showed rather high titers: by 8-10 days, 2.5x10 ² and 3.9x10 ^2, respectively.

 A cytological analysis of the purulent wound showed that on the 4-6 day of treatment with Abisil-1, obtained according to example 2, there is a significant number of neutrophils (up to 70%); the appearance of macrophages and lymphocytes, fibroblasts is noted in the spectrum of cells; some microbes are located intracellularly. In the case of using the Fusidin gel in cytograms, a large number of destroyed neutrophils are observed. A significant number of microbial cells are located extracellularly and intracellularly.

 On the 8th day of treatment with Abisil-1 in the cytogram, the content of neutrophils decreased to 50-60%, the number of macrophages increased. There are few microbial cells, and they are in a state of phagocytosis. In the control group (untreated) by this time there is a large number of neutrophils and macrophages in degenerated and phagocytic forms, single fibroblasts; a large number of microorganisms are located extracellularly. Using the Fusidin gel, a large number of neutrophils is noted in smears, up to 75% of 5-10% of the cellular composition is accounted for by lymphocytes, monocytes, macrophages and fibroblasts.

 By the 12th day of treatment, the imprints of the group of animals treated with Abisil-1 showed a further decrease in the level of neutrophils (up to 70%). Microbial cells are practically absent. A similar picture in the groups of animals using the gel is noted by 17 days (Fusidin gel) and 22-24 days in the control (untreated) group of animals.

EXAMPLE EXAMPLE 3. The method is carried out as in Example 1 except that extracted from the capsular extract of Siberian fir fraction of essential oils comprising monoterpene compound is mixed with a capsular extract ^at 20 ° C in the ratio 1: 99 (1%) , followed by dissolution in vegetable (sunflower) oil in a ratio of 1: 4 (20%). On the 2nd day of treatment of animals with Abisil-1, obtained by the specified method, the clinical picture corresponded to that which was noted using the means of example 2.

 A significant difference appeared from 12-17 days of observation. By 12 days, the bottom of the wound has not yet been completely filled with granulation tissue. On the periphery of the wound, weaker areas of marginal epithelization appeared.

 Granulation tissue becomes completely fibrosed by 16-17 days. The wound becomes completely epithelized by 18-19 days. By this time, a scar forms, and along the edges of the wound, the growth of the coat.

 Anti-inflammatory and wound healing activity, reflected in the table. 2 and 3, shows that by 17 days the percentage of wound surface healing is 94.3 ± 0.1 versus 53.3 ± 5.1 in the control and 78.7 ± 6.1 of the Fusidin gel.

A study of the dynamics of inoculation revealed a low titer of microbial bodies by 6 days: 1.4 x 10 ⁴ versus 4.8x10 ⁶ controls and 3.5x10 ⁴ Fusidin gel.

 Cytological analysis of wounds on the 12th day of treatment of animals with Abisil-1, obtained according to example 13, showed that profibroblasts, polyblasts and macrophages predominate in the prints. The number of neutrophils decreased to 44%. Microbial cells are absent.

 PRI me R 4. Patient S. 33 years. Diagnosis of the underlying disease erysipelas of the right thigh; a neurological bullous form, epifascial phlegmon of the right lower limb.

Before treatment with Abisil-1 was started, Levomikol ointment and surgery were used: autopsy, debridement, phlegmon drainage. No improvement came. The body temperature from subfebrile increased to 37.8 ^about C.

 A day after the start of treatment with Abisil-1, the pain ceased to bother the patient; the condition has improved; Intensive granulation of the wound began; the temperature returned to normal. By 5 days, bright granulations were observed; swelling began to subside. By 10 days, the wound was almost cleaned against granulation and epithelization.

 The effectiveness of using "Abisil-1" is high. Side effects were not identified.

 PRI me R 5. Patient P. 42 years. The diagnosis of underlying disease is post-infection encephalopathy.

 Before the start of treatment with Abisil-1, Vishnevsky ointment was used. Patient's condition: moderate.

 After the start of the use of the drug "Abisil-1", the patient improved sleep, appetite, general condition; rapid wound cleansing, granulation and epithelization occurred. Edema by 5 days was practically absent; sore wounds were not noted; hypergranulation of the wound was noted.

 The effectiveness of using the drug "Abisil-1" is high. Side effects were not identified.

 PRI me R 6. Patient M. 36 years. Diagnosis: blunt trauma of the abdomen, rupture of the small intestine. Spilled fibro-purulent peritonitis. Phlegmon of the anterior abdominal wall. The patient underwent surgery: suturing a rupture of the small intestine, debridement, drainage of the abdominal cavity. Programmed Laparatomy. Used: antibiotics, ointment "Levomikol". The patient's condition is serious. Since the beginning of the use of the drug "Abisil-1", a decrease in temperature, improvement in general condition, a decrease in pus content, and a length of the wound decrease. By 2 days, marked gruntation is noted.

 The effectiveness of using the drug is high: there comes a quick cleansing, granulation and epithelization of tissues. Side effects were not identified.

 Thus, the study of comparative anti-inflammatory and wound healing activity, based on the analysis of the clinical picture of the inflammatory process and repair of wounds, the dynamics of seeding microbes from purulent wounds, the period of wound healing and a cytological study of the course of the wound process showed that the proposed tool Abisil-1 (oil solution of the extract Siberian fir capsules, enriched in a fraction containing monoterpene compounds in a ratio of 19.5: 0.1: ost-e), exhibits a large anti-inflammatory and wound vivifying activity (97.3 ± 3.4% of wound healing by 17 days) than means containing ratios of 19.5: 0.02: ost and 19.5: 0.2: ost-fractions of monoterpene compounds ( 90.5 ± 94.3 ± 0.1), as well as Fusidin gel (78.7 ± 6.1) or control (53.3 ± 5.1). In addition, Abisil-1 with a period of use of 7-10 days causes a quick cleansing of the wound from purulent-necrotic masses, slows down exudative and stimulates reparative processes, i.e. is a drug that allows it to be used in phases I and II of the wound healing process, which is not typical for known drugs of a similar pharmacological action.

 As a means of comparison, a natural capsular extract of Siberian fir was not used, because a decrease in enrichment from 0.1% to 0.2% with monoterpene compounds significantly decreased the activity threshold (see Tables 2 and 3).

 The fraction of monoterpenic compounds did not seem to be studied as such because of its high toxicity, and also because its increase to 0.2% in the resulting product did not cause a surge in increased activity.

 Thus, the introduction of a Siberian group fraction containing monoterpene compounds into the capsule extract of fir obtained as a result of fractional separation of the capsule extract and dissolution of the resulting complex in vegetable oil allows one to obtain an agent with high anti-inflammatory, antibacterial, and wound healing activity.

Claims (1)

An agent with anti-inflammatory, antibacterial and wound healing activity based on a plant component, characterized in that it contains a capsular extract of Siberian fir Abies sibiricus Zedebur, monoterpene compounds of essential oils and vegetable oil in the following ratio, wt.%:
Capsule extract of Siberian fir - 19.0 - 20.0
Monoterpene compounds of essential oils - 0.02 - 0.20
Vegetable Oil - Else

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