BRPI1100760A2 - SUSTAINED RELEASE BIOCURATIVE OBTAINED FROM BIOCELLULOSIS AND PROPOLIS EXTRACT, AS WELL AS THE PROCESS FOR OBTAINING THERAPEUTIC APPLICATIONS - Google Patents

Abstract

SUSTAINED RELEASE BIOCURATIVE OBTAINED FROM BIOCELLULOSE AND PROPOLIS EXTRACT, AS WELL AS ITS PROCESS OF OBTAINING AND THERAPEUTIC APPLICATIONS The present invention relates to biocuratives consisting of biological membranes, containing different concentrations of Propolis Extract, associated or not with vegetable derivatives of Melaleuca (Melaleuca alternifolia) or Rosemary (Rosmarinus officinalis) or Thyme (Thymus vuIgaris,) or other natural derivatives of interest, and such biocuratives have the following activities: antimicrobial, against gram positive and gram negative microorganisms, healing, anti-inflammatory, analgesic and anesthetic, besides offering tissue material reconstitution (reepithelization), obtained by a relatively simple and low-cost production process, which aims to: assist in the treatment of wounds, skin lesions, burns, chronic wounds, including patients diabetics, stimulate scarring will surgeries, including plastic, aesthetic nature or not, among other pathologies involving the antiseptic activity, healing, regenerating tissue, inflammatory, analgesic and anesthetic.

Description

SUSTAINED RELEASE BIOCURATIVE OBTAINED FROM BIOCELLULOSIS AND PROPOLIS EXTRACT, AS WELL AS THE PROCESS FOR OBTAINING AND THERAPEUTIC APPLICATIONS FIELD OF THE INVENTION

The present invention relates to bacterial cellulose antimicrobial and healing membranes containing propolis extract as active substance, whether or not associated with plant derivatives or other products of natural origin, and its application as a temporary skin substitute in the treatment of burns. , skin or tissue lesions, chronic wounds and wounds in general, ie an unprecedented, high-performance biocurative with characteristics suitable for induction of the healing process, local asepsis, anti-inflammatory and analgesic action, with surprising advantages over conventional treatments. , whose application is not restricted to the pharmaceutical field, having diverse applications in medicine (in its various specialties), dentistry, veterinary, agronomy, medical or surgical materials, etc. BACKGROUND OF THE INVENTION Several studies seek new forms of treatment for

regeneration of skin lesions, as it is known that classic wound treatments protect the lesion but do not promote its healing. It is further known that in conventional treatments, repeated administration of the dosage forms is necessary to achieve therapeutic success, which is impractical and very painful. Thus, a sustained release system capable of releasing the drug into the wound bed gradually offers numerous advantages over conventional systems, especially in injured, injured or burned areas, as the single application of the biocurative avoids manipulation, pain. and possibility of contamination of the area, maintaining optimal therapeutic levels with minimum fluctuations. Currently, we seek to obtain products that reduce healing time and contamination, promoting pain relief, a situation in which the present invention is successful.

In Brazil, according to the Brazilian Society of Burns, one million cases of burns occur each year, 200,000 are treated in emergency services, and 40,000 require hospitalization. Burns are among the leading external causes of death in Brazil, second only to other violent causes, including traffic accidents and homicides.

Healthy skin is the first and main barrier against bacterial invasion, but in burned patients the skin is destroyed, and consequently the underlying exposed tissue is an excellent means for bacterial development. The occurrence of infections is therefore one of the major problems in burns, emphasizing that septicemia is involved in about 50% of deaths from burn injuries.

With regard to the treatment of wounds (burns, varicose ulcers, graft donor areas) a major advance is still needed today to minimize patient suffering.

The classic treatments of wounds (bandages, gauze,

example) protect the lesion, but do not promote healing, and generate excellent conditions for germ proliferation, requiring frequent changes, thus impairing the healing process. The aim is to produce new dressings that provide, besides wound protection, a therapeutic effect that assists in the healing process.

The key features of an ideal skin substitute are: adhesion, water vapor permeability, elasticity, durability, barrier to bacteria, non-antigenic, hemostatic in action, easy to apply and handle, and ultimately low cost (WOUK et al., 1998). Currently the treatment of chronic wounds has been performed by different types of materials (hydrogels, hydrocolloids, synthetic or biological membranes), such as: Epicel® - Genzyme Biosurgey (US $ 18.90 / cm2), Alloderm® - LifeCeII Inc. (US $ 10.60 / cm2), Apligraf® - Organogenesis Inc. ($ 25.60 / cm2), Biobrane® - Mylan Lab. ($ 0.90 / cm2), Integra® - Integra LifeScience Gorp. (US $ 6.00 / cm2), of which the first three are related to cadaver dermis and bovine collagen added to keratinocytes and fibroblasts, and the last two are of synthetic origin, such as (i) nylon and silicone and ( ii) glycosaminoglycans and silicone. Imported biomaterials, which make the treatment process more expensive, and many of them have low adhesion to the wound bed, and also require frequent changes, as well as the possibility of contamination.

In addition to the above, silver-based compounds are used for such treatments as silver sulfadiazine-based ointments and creams in addition to nanoparticulate silver, as noted in patent application PI0513967-8. Although the results of these materials are satisfactory, a limitation occurs because it works with metal particles that may be toxic to the human organism. Bacterial cellulose (CB) membranes / films have emerged as the most efficient dressing, as it has superior properties when compared to the others, besides presenting an extremely lower cost. The advantages of using CB film as a biological dressing have been confirmed in over 300 treatments (CZAJA, et al., 2007).

Biocellulose membranes can be obtained by biosynthesis of Acetobacter xylinum bacteria in static culture medium that have glucose as a nutritional source. CB membranes are obtained as a highly hydrated hydrogel (99% water), being chemically pure (free of lignin, hemicellulose, and pectins). It has nanometric cellulose chains, organized in a three-dimensional structural arrangement, which generates a highly crystalline system (60-80%) with excellent mechanical strength. This network of nanometric yarns gives it a huge surface area, amazing water absorption and retention capacity, good elasticity, and is easily molded, desired characteristics for an ideal dressing. It is a biodegradable, biocompatible, non-toxic and non-allergenic product. Although bacterial cellulose acts as a dressing in the

healing of chronic wounds and burns, its application to contaminated wounds is limited as it has no antimicrobial activity.

However, as CB is obtained "in natura" as a highly swollen and porous hydrogel, one possibility is to insert organic / inorganic compounds into its structure to produce the desired activity.

In order to produce a biocutative that acts as effectively as other materials previously developed, and with the advantage of having its natural origin, one possibility is the incorporation of propolis to CB membranes, producing a new biocurative.

Currently over 300 propolis constituents have been identified, most of which are composed of polyphenols such as flavonoids, phenolic acids and esters, aldehydes, ketones and others. Propolis has been used since ancient times. In general, it is made by bees from bud resins, flower buds, and other parts of plant tissues. Crude propolis is in the solid state at room temperature and its physical properties vary from one sample to another, implying a high variation of its active principles and presenting a varied and complex chemical composition, depending on where the propolis is harvested. It is known that the antimicrobial and immunomodulatory properties of propolis do not vary with their harvesting time, however it is important to know about the concentration of the active ingredients and their standardization for the preparation of a pharmaceutical form whose active ingredient is of vegetable origin. . Propolis compounds with pharmacological properties include flavonoids (flavones, flavonols and flavonones), which are characterized by having in their chemical structures at least one aromatic ring directly linked to one or more hydroxyls. Its properties are partly explained by its ability to protect against the aggressive action of free radicals. For decades several biological activities have been attributed to propolis, highlighting the antibacterial, antiviral, antifungal, anti-inflammatory activity. anticarcinogenic, anesthetic, cytostatic, immunostimulant and healing. Therefore, propolis bioactive compounds are bacteriostatic agents, as they decrease the growth of pathogenic and bactericidal microorganisms, since in many cases they kill such microorganisms, as well as their penetration in the body, avoiding invasive infections and delaying the healing process of burns. . Propolis is a good alternative for antimicrobial use in burns, as it has no acute toxicity and its compounds are biodegradable, a factor that is important when talking about environmental issues.

Therefore, combining the antimicrobial properties of propolis with dressings proves to be an interesting way to treat pressure ulcers, injuries, wounds and burns. A water-soluble propolis-impregnated dressing is already on the market, which is marketed as non-adherent gauze. Its indication is for superficial or deep second degree burns, for use in donor and recipient areas of dermo-cutaneous grafts and in traumatic injuries, but the present proposal is much more powerful due to the absorption capacity of biomembranes in relation to the extract. propolis, to the characteristics of the biocurative in the injured tissue, since this membrane has flexibility, adherence, is permeable to oxygen giving all the ideal conditions for the stimulation of tissue repithelization, besides possessing in addition to the characteristics of the aforementioned biomembranes, the impressive properties propolis, namely its antimicrobial, healing, anti-inflammatory, analgesic and anesthetic activity, among others that do not apply to the present case.

The method of applying these dressings follows a standard procedure that comprises the following steps: a) asepsis of the lesion site with antiseptic;

b) removal of devitalized tissue;

c) dressing position over the injured area;

d) remove air bubbles and excess blood with saline soaked gauze to increase

skin dressing;

e) dressing application;

However, conventional dressings should be replaced every 12 or 24 hours due to the amount of secretion and exudate produced. Therefore, they have disadvantages as to the application method and the required frequency of dressing change, which does not happen in the present invention, as it does not require replacement, it is characterized as a sustained propolis release system, thus maintaining optimal levels of propolis. active ingredients in the first hours of treatment, besides being permeable and not maintaining excess exudates in the region, which hinder the healing process.

There is a body of research in the literature involving biocuratives and other pharmaceutical forms, and such research reflects the current importance of finding ways to prevent microbial proliferation in infectious processes or as prophylactic aseptic measures. Currently, for the treatment of chronic wounds, it is worth mentioning nitrofurasone and silver sulfadiazine, the latter having a broad spectrum of action.

However, both drugs may cause adverse reactions and have some disadvantages. Nitrofurasone, for example, causes allergic contact dermatitis and may have its effect reduced in the presence of blood and organic matter. Sulfadiazine, on the other hand, may cause a burning sensation, hives, itching and skin necrosis. Due to its low solubility in water, its absorption is also limited.

In this sense, there remains a need for a biocurative that has antimicrobial action, which would prevent the formation of infected biofilms when bacteria are adhered to the skin surface; It has characteristics that are close to the skin, which is efficient, which accelerates the tissue regeneration process and requires a lower frequency of exchange. Biofilm is a metabolic load imposed by bacteria on the wound bed. In addition to competing with normal cells for oxygen and nutrients, the presence of biofilm compromises and identifies various stages of the wound healing process. Hence the importance of avoiding their formation in regenerative processes. On the other hand, some patent documents disclose propolis-based compositions impregnated with textile materials or wound healing tapes, as can be seen from RU2319487 and CN2912647. However, contrary to the present invention, these documents do not use bacterial cellulose in conjunction with propolis, the combination of which is effective in treating and healing skin or tissue injuries such as burns, pressure ulcers, chronic wounds, among others. others without the proliferation of 10 bacteria. The advantage of using bacterial cellulose is its transparent appearance and the need for no dressing change, as well as its sustained release.

OBJECTIVES OF THE INVENTION

More specifically, the present invention is characterized by bio-curatives consisting of biological membranes, preferably bacterial cellulose membranes, containing different concentrations of Propolis Extract, preferably standardized propolis extract (EPP-AF®), whether or not associated with plant derivatives. Melaleuca (Melaleuca alternifolia) or 20 Rosemary (Rosmarinus officinalis) or Thymus (Thymus vulgaris) or other natural derivatives of interest, and such biocuratives have the activities: (i) antimicrobial, against gram positive and gram negative microorganisms, (ii ) healing, (iii) antiinflammatory, (iv) analgesic and anesthetic, besides offering tissue material reconstitution (reepithelization), obtained by a relatively simple and low cost production process, which aims to: assist in treatment of wounds, les skin lesions, burns, chronic wounds, including diabetic patients, stimulate the healing of surgeries, including plastic, aesthetic or not, among other conditions involving antiseptic, healing, regenerative of 10 tissues, anti-inflammatory, analgesic and anesthetic , as previously informed. The present invention is not restricted to the pharmaceutical field, having diverse applications in medicine (in its various specialties), dentistry, veterinary, agronomy, medical or surgical materials, etc.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows the Infrared Vibrational Spectroscopy of: a) Propolis; b) CB and c) Hydrated CB containing propolis.

Figure 2 Illustrates the X-ray Diffractogram of: a) CB; b) hydrated CB containing 1.2% propolis; b) hydrated CB containing 2.4% propolis; b) Hydrated CB containing 3.6% propolis. Figure 3 shows the Scanning Electron Microscopy of: (a) CB; (b) CB containing propolis.

Figure 4 illustrates p-coumaric chemical marker.

Figure 5 illustrates the isosakuranetina chemical marker.

Figure 6 illustrates the chemical marker artepelin c.

DESCRIPTION OF THE INVENTION

The invention relates to antimicrobial biological membranes and

t

preferably bacterial cellulose containing propolis extract as the active substance, preferably standardized propolis extract (EPP-AF®), whether or not associated with plant derivatives or other products of natural origin. skin treatment in the treatment of burns, skin or tissue injuries, chronic wounds and wounds in general, ie an unprecedented high-performance biocurative with characteristics suitable for induction of the healing process, local asepsis, anti-inflammatory and analgesic action , with surprising advantages over conventional treatments.

The process of obtaining the invention now required is characterized by the following steps:

The. Active Input: Propolis Extract, preferably Standard Propolis Extract (EPP-AF®); B. Solubilization of tea tree or rosemary or thymus oil or any other derivative of plant or natural origin that may add activity of interest to the product (in a concentration that may vary from 0.1 to 99%, as needed), in the standardized extract of

propolis previously presented, with the aid of some adjuvant, if necessary (emulsifier, solubilizer, or other);

ç. For bacterial cellulose production, the following procedure follows: Acetobacter xylinum cultures are grown at a cultivation time of 24-140 hours at a temperature of 25 to 30 ° C.

0C. Culture medium (Schramm-Hestrin medium) has the following basic composition: glucose 2% (w / v), peptone 0.5% (w / v), yeast extract 0.5% (w / v), phosphate anhydrous disodium 0.27% (w / v) and citric acid monohydrate 0.115% (w / v).

d. Preparation of biocuratives:

(i) The CB membrane is removed from the culture medium and

properly treated with strong bases for bacteria removal. Hydrated CB samples are thoroughly washed for inclusion of Propolis Extract, preferably standard propolis extract (EPP-AF®) associated with plant derivative.

selected.

(ii) Bacterial cellulose membranes (hydrated or dried) are immersed in Propolis Extract solutions, preferably standardized propolis extract (EPP-AF®), associated with the plant drug derivative at concentrations from 0.1 to 99%. (w / w);

(iii) Maintaining the solution in contact with the membrane for 12 to 48 hours at room temperature (20 to 30 ° C);

(iv) Propolis-containing CB membranes are dehydrated

at a temperature of 20 to 40 ° C until a bacterial cellulose film containing flexible, supple amber-colored propolis extract (light or dark) is obtained depending on the concentration of propolis.

(v) Next the propolis-containing CB membranes are

stored in properly dried containers at room temperature.

In the bacterial cellulose membrane of the present invention, the antimicrobial action of the biocurative is promoted by the presence of the 15 bioactive substances of propolis and the plant drug derivative used, according to the chemical profile and standardization of propolis compounds as described in patent (PI0405483). -0). When in contact with the wound, the biocurative sustained release (Figure 3) into the wound bed of the bioactive compounds 20 contained in the interstices of the three-dimensional cellulose web. The bacterial cellulose membrane should be previously moistened in saline, preferably for topical use in chronic wounds and burns.

It is important to note that the use of propolis and plant drug derivatives has numerous advantages, as they are not toxic and do not induce microbial resistance, unlike conventional antibiotics. Due to the widespread and indiscriminate prescription of antibiotics, pathogens have become increasingly resistant to the actions of such agents. Propolis and plant derivatives are therefore very useful prophylactic and therapeutic agents in the prevention of wound colonization by microorganisms (gram positive and gram negative) which, when present in wounds, injuries or burns, delay the healing process and, therefore the cure.

Since the bacterial cellulose membrane is a biocompatible membrane, it also has properties of accelerating tissue regeneration and healing. By being transparent, it is possible to follow the evolution of the healing process without removing the dressing. Despite the concentration of propolis used, the more concentrated the propolis solution employed, the more intense is the characteristic amber color of the biocurative, however it remains with good transparency, allowing the evaluation of the healing process without removing it from the applied site. One of the advantages of these biocuratives is that they have sustained release, once in the skin, do not need to be changed, unlike the dressings currently used. Throughout the healing process, the CB membrane containing propolis 5 remains on the skin and as tissue regeneration occurs, the skin surface detaches. Moreover, in cases of first, second and third degree burns, the application of the propolis-containing CB membrane promotes pain relief, favors hemostasis, does not allow water loss, forms a barrier against external agents and has a blocking action on the propellants. UVA and UVB rays.

These properties make the membrane useful not only in the treatment of chronic wounds and burns, but also in other conditions involving difficulty in healing and tissue regeneration, such as diabetes, graft donor areas, and others. In elderly patients, the present invention has been shown to have an important effect, as such patients have greater physiological difficulty in the healing process due to advanced age.

EXAMPLES

EXAMPLE 1 - Bacterial Cellulose Biocuratives Containing

Propolis Extract and / or derivative of plant or natural origin The biocuratives were obtained as described below:

The. Active Input: Propolis Extract, preferably standardized propolis extract (EPP-AF®), characterized chemically and biologically as described in patent process (PI0405483-

0), in various concentrations and dilutions. The propolis extract used was supplied by Apis Flora Indl. I ate. Ltda., Which was obtained after a careful study of the raw material from different regions of Brazil;

B. Solubilization of tea tree oil (Melaleuca alternifolia) or rosemary (Rosmarinus officinalis) or thymus (Thymus vulgaris) or

Aloe vera or another derivative of plant or natural origin in the standardized propolis extract previously presented (in order to improve the performance of propolis biocuratives or to add other biological activities to it);

ç. For bacterial cellulose production, the following is as follows:

Procedure: Acetobacter xylinum cultures are grown at a cultivation time of 24-140 hours at a temperature of 25 to 30 ° C. The culture medium (Schramm-Hestrin medium) has the following basic composition: glucose 2% (w / v), peptone 0.5% (w / v), yeast extract 0.5% (w / v), anhydrous disodium phosphate 0.27% (w / v) and citric acid monohydrate 0.115% (w / v);

d. Preparation of biocuratives: (i) The CB membrane is removed from the culture medium and properly treated with strong bases for bacterial removal. Hydrated CB samples are thoroughly washed for inclusion of the standardized Propolis Extract (EPP-AF).

to the selected vegetable derivative.

(ii) Bacterial cellulose membranes (hydrated or dried) are immersed in Propolis Extract solutions, preferably standardized propolis extract (EPP-AF®), associated with the plant drug derivative at concentrations from 0.1 to 99%. (w / w);

(iii) Keep the solution in contact with the membrane during

12 to 48 hours at room temperature (20 to 30 ° C);

(iv) Propolis-containing CB membranes are dehydrated at a temperature of 20 to 40 ° C until a bacterial cellulose film containing propolis-like extract is obtained.

malleable, flexible amber (light or dark) color depending on the concentration of propolis.

(v) Thereafter the propolis-containing CB membranes are stored in properly dried containers at room temperature;

EXAMPLE 2 - Chemical Characterization of Biocuratives -

bacterial cellulose membranes containing Propolis Extract

The biocuratives (composites) described in example 1 were characterized by infrared vibrational spectroscopy (FT-IR), x-ray diffractometry (XRD) 1 scanning electron microscopy (SEM) 1 among others. Vibrational Infrared Spectroscopy allows qualitative identification of functional groups present in the developed composites. Figure 1 shows representative Infrared spectra for propolis, CB and a hydrated CB membrane containing propolis, respectively. Some bands that characterize propolis in the IR spectrum are absorptions referring to the presence of double bond C = C (vmax 1641 cm-1), carboxyl (vmax 1703 cm-1) and hydroxyl (vmax 3487 cm-1) groups. . The main attributions that characterize CB are: 3500 cm-1: OH stretch; 2900 cm-1: CH alkane stretch and asymmetric stretch CH2; 2700 cm-1: symmetrical stretch CH2; 15 1640 cm-1: OH strain; 1400 cm-1: CH2 strain; 1370 cm-1: CH3 strain; 1340 cm-1: OH strain and 1320-1030 cm-1: CO strain. Basically, the CB membranes containing propolis have spectra that can be considered as the sum of the CB spectrum and propolis, showing the formation of a composite material. The band located around 1703 cm -1 characteristic of carboxyl groups (vmax 1703 cm-1), present exclusively in propolis, is clearly observed in the membranes of propolis-containing CB. Figure 2 shows the X-ray diffractogram for CB and CB containing propolis The peaks observed at 15 and 22.5 0C refer to CB, referring to the contributions of phases 1a and 1β It can be inferred that due to the presence of propolis in CB, there is a change in the diffractogram profile, or ie, a gradual decrease in the crystallinity of CB with increasing propolis concentration Figures 3 (a) and (b) show results of Scanning Electron Microscopy (SEM) for CB and CB membrane with Figure 3 (a) shows the surface morphology of the CB, which is characterized by a compact mesh formed by a tightly interwoven web of long nanometer-sized fibers (“nanocelluloses”). Figure 3 (b) for propolis-containing CB shows the homogeneous coating of CB fibers, which can be attributed to the presence of propolis. The characterization of the qualitative and quantitative chemical profile of propolis in biocuratives is not presented in this application because it has already been described in the patent application of Standardized Propolis Extract (EPP-AF®) (PI 0405483-0 - Published in Industrial Property Magazine no. 1778 of 02/01/2005) - active input of the present biocuratives. EXAMPLE 2 - Bacterial Cellulose Biocuratives Containing Standardized Sustained Release Propolis Extract (CB-propolis)

For the evaluation of the release profile of the standardized propolis extract (EPP-AF®) of the biocuratives, a Sotax AT7 smart Dissolver was used, equipped with the 5 - shovel on disk apparatus. of drugs in transdermal patches. For the dissolution medium the temperature indicated in the literature is 32 ± 0.5 0C1 pH 5.0 at 6.0 and blade rotation at 100 rpm. (SIEWERT et al, 2003). A minimum volume of medium was also stipulated for the vat, which is 500 ml. According to the sink conditions determined from the solubility of the propolis extract used in the production of biomembranes and the minimum volume of 500.0 mL in each vat, 50% ethanol / phosphate buffer pH 6.0 medium was used. The medium was degassed immediately after preparation, so that air bubbles did not interfere with the dissolution rate, thus altering the results. Samples from all biomembranes were cut to 4.0 cm in diameter and adapted to the apparatus to remain with a release surface in contact with the medium. During the dissolution test, 5.0 ml aliquots were removed with the aid of a 5.0 ml graduated pipette at the following times: 10min, 30min, 1h, 2h, 3h, 6h and 24h. Always remember to add the same amount of medium immediately after withdrawal to keep the volume constant. For analysis of the samples in high performance liquid chromatography, aliquots were prepared to obtain solutions with optimal concentrations for injection in HPLC. The results obtained are shown in figures 4, 5 and 6 and demonstrate, through the markers chosen for this assay, namely p-coumaric acid (polar compound), isosakuranetin (flavonoid) and artepellin C (support compound) that the biocuratives Independent of the propolis concentrations of the composites, they present sustained release, demonstrating that the biocuratives do not need to be reapplied, that is, they have the immense advantage of not having to change, avoiding inconvenience for the patients and pain relief.

EXAMPLE 3 - Antimicrobial Activity of CB-Propolis Biocuratives

Studies show that microorganisms such as Spaphylococcus aureus, Pseudomonas aeruginosa and Enterobacteria are important pathogens in surgical wound and burn infections. Among these microorganisms, the antimicrobial activity of CB-propolis membranes in strains of Staphylococcus aureus - ATCC 25923, S. aureus MRSA (methicillin resistant) ATCC 43300 and Staphyloeoeeus epidermidis ATCC 14990, figure 4, were evaluated. to bacterial cellulose biocuratives containing Standard Propolis Extract (EPP-AF®) at the propolis concentrations contained in the biocuratives and inhibition halos were demonstrated demonstrating the activity of the biocuratives in these systems. The evaluation of the antimicrobial activity of biocuratives was performed in duplicate using the CLSI 10 agar diffusion method (NCCLS, 2003b) against the following microorganisms: S. aureus ATCC 25923, S. aureus (MRSA) ATCC 43300, S epidermidis ATCC 14990, K. pneumoniae ATCC 10031, P. aeruginosa ATCC 27853 and E. eoli ATCC 25922. A suspension of each microorganism (24 hour culture) was prepared in 15 0.85% saline with scale equivalent turbidity. 0.5 McFarIand (approximately 1,108 CFU / ml). The suspensions were measured in a spectrophotometer at a wavelength of 625nm considering the absorbances between 0.08 and 0.1. With the aid of a sterile cotton swab, each suspension was seeded in Petri dishes (90x15mm) containing Mueller Hinton agar. The disc-cut biocuratives were distributed at equidistant points on the surface of the Mueller Hinton agar. Then 5μΙ of sterile 0.85% physiological solution was added to the surface of each biocurative. The plates were incubated at 36 ° C for 18 hours. After the incubation period, the inhibition halos were measured with the aid of a 5 millimeter ruler. In addition, the study was also conducted for some other plant derivatives, such as melaleuca (Melaleuca alternifolia), pomegranate (Punica granatum) and barbatimão, showing that the melaleuca has powerful action against gram negative microorganisms, while the others show activity against 10. gram positive.

EXAMPLE 4 - CB-Propolis Healing Activity Standardized Propolis Extract (EPP-AF®) has been extensively studied for its biological activities, such as antioxidant, antimicrobial, healing, anti-inflammatory, analgesic and anesthetic, antiviral, etc. as already described in the Standardized Propolis Extract (EPP-AF®) patent, active ingredient of the present invention (PI 0405483-0 - Published in Industrial Property Magazine No. 1778 of 02/01/2005), in addition to the data already presented. in the patent for the same active ingredient in a particular application of the invention: "Sustained-release heat-reversing gel containing standardized propolis extract (EPP-AF®) and its manufacturing process, with antiseptic and healing activity for treatment of burns, injuries of skin and others ”, where the application regarding the induction of healing of the active ingredient is demonstrated.

Claims (10)

1. Process, characterized in that it comprises the following steps: a. Active Input: Propolis Extract, preferably standard propolis extract (EPP-AF®); b Inclusion of tea tree oil or rosemary or thymus in the propolis extract process; ç. For bacterial cellulose production, the following procedure is followed: Acetobacter xylinum cultures are grown at a cultivation time of 12-140 hours at a temperature of 25 to 30 ， ， where the culture medium (Schramm-Hestrin medium) has the following basic composition: glucose 2% (w / v), peptone 0.5% (w / v), yeast extract 0.5% (w / v), anhydrous disodium phosphate 0.27% (w / v) and citric acid monohydrate 0.115% (w / v); d. Preparation of the biocuratives: (i) The CB membrane is removed from the culture medium and properly treated, properly treated with strong bases for bacterial removal, and hydrated CB samples are thoroughly washed to include Propolis Extract associated with the selected plant derivative. ; (jj) Bacterial cellulose membranes (hydrated or dried) are immersed in plant drug derivative propolis extract solutions at concentrations of 0.1 to 99% (w / w); (jjj) Maintain the solution in contact with the membrane for 12 to 48 hours at room temperature (20 to 30 ° C); (iv) Propolis-containing CB membranes are dehydrated at a temperature of 20 to 40 ° C until a bacterial cellulose film containing flexible, supple amber-colored (light or dark) propolis extract is obtained depending on the concentration of propolis; (v) Thereafter the propolis-containing CB membranes are stored in properly dried containers at room temperature; Biocurative, characterized in that it consists of biological membranes, preferably bacterial cellulose membranes, containing different concentrations of propolis extract, preferably standardized propolis extract (EPP-AF®); whether or not associated with plant derivatives of Melaleuca (Melaleuca alternifolia) or Rosemary (Rosmarinus officinalis) or Thymus (Thymus vulgaris) in concentrations from 0.1 to 99% (w / w); and such biocuratives present antimicrobial activities, against gram positive and gram negative microorganisms, healing, anti-inflammatory, analgesic and anesthetic, besides offering full reconstitution of tissue material. Biocurative according to Claim 2, characterized in that the antimicrobial action of the biocurative is promoted by the presence of propolis bioactive substances and the plant drug derivative used. Biocurative according to Claim 2, characterized in that the biocurative sustained release into the wound bed of the bioactive compounds contained in the interstices of the three-dimensional cellulose web. Biocurative according to Claim 2, characterized in that the bacterial cellulose membrane is a biocompatible membrane, having accelerating properties, tissue regeneration and healing. Biocurative according to Claim 5, characterized in that the bacterial ceiosis membrane is transparent and, depending on the concentration of the propolis solution employed, is amber in color. Biocurative according to Claim 6, characterized in that the healing takes place without the removal or exchange of the biocurative from the applied site. Biocurative according to Claim 2, characterized in that it has sustained release. Biocurative according to Claim 2, characterized in that it promotes pain relief, favors hemostasis, and does not allow water loss; Biocurative according to Claim 2, characterized in that it forms a barrier against external agents with blocking action on UVA and UVB rays.