BRPI0804288A2 - lippia salviaefolia cham. extracts, active fractions, compositions and their use - Google Patents

Abstract

LIPPIA SALVIAEFOLIA CHAM. EXTRACTS, ACTIVE FRACTIONS, COMPOSITIONS, AND THEIR USE. The present invention relates to Lippia salviaefolia cham. Leaf extracts, their active fractions, pharmaceutical and / or cosmetic compositions and their use as a natural antimicrobial (preservative).

Description

"LIPPIA SALVIAEFOLIA CHAM. EXTRACTS, ACTIVE FRACTIONS, COMPOSITIONS AND THEIR USE"

Field of the Invention

The present invention relates to Lipia salviaefolia Cham. Leaf extracts, their active fractions, pharmaceutical and / or cosmetic compositions and their use as a natural (conseryante) antimicrobial.

Background of the Invention

The genus Lippia (Verbenaceae) includes approximately 200 species of herbs, shrubs and small trees. These species are mainly distributed by the countries of South America and Central America, and the tropical territories of Africa (TERBLANCHE, FC & KORNELIUS, G., Essential oil constituents of the Lippia genus (Verbenaceae). , 471-485, 1996). Most species of the genus Lippia are traditionally used to treat gastrointestinal and respiratory disorders. Some species have demonstrated antimalarial, antiviral, and cytostatic activity. (MORTON In: Atlas of Medicinal Plants of Middle America I, Springfield, Illinois, USA (1981), pp. 745-750; GASQUET, M., DELMAS, F., TIMÓN-D AVI D., P., KEITA, A., GUINDO, M. KOITA, N., DIALLO, D., DOUMBO, O., In vitro and in vivo evaluation of a traditional antimalarial, "Malarial-5". 423-427, 1993; ABAD, MJ, SANCHEZ, S., BERMEJO, P., VILLAR, A., CARRASCO, L., Antiviral activity of somemedicinal plants.Met. Find., 17 (Suppl. A) p.108 , 1995). In general, the genus has a consistent profile of chemical composition, pharmacological activities and traditional use. Two of the most important species of this genus are L. multiflora and L. alba (PASCUAL, ME, SLOWING, K., CARRETEO, E., SÁNCHEZ MATA, D., VILLAR, A., Lippia: Traditional uses, chemistry and Pharmacology: a J. of Ethnopharmacol 76, Issue 3, 201-214, 2001). In the literature, there is a record of other Lippia species with antimicrobial activity such as Lippia sidoides (COSTA, S. M. O .; LEMOS, T. L.

G.; PESSOA, O. D. L .; ASSUNÇÃO, J. C. C; BRAZ-FILHO, R .; 2002.Chemical constituents of Lippia sidoides (Cham.) Verbenaceae. Rev.Bras. from Farmacog., v. 12, suppl., P. 66-67.) And Lippia adoensis (TAGET,

H. ; MOHAMMED, E .; ASRES, K. GEBRE-MARIAM, T., Antimicrobialactives of some selected traditional Ethiopian medicinal plants used for the treatment of skin disorders. J. of Ethnopharmacol. 100, p. 168-175,2005), among others. However, there are no references in the literature on pharmacological activity, toxicity studies or traditional use of the L. saluiaefolia species.

L. salviaefolia is a plant originally from Brazil, belongs to the large group of Dicotyledons, family of Verbenaceae. As a vegetative habit, it is characterized as shrub with aromatic leaves, yellowish corolla tube and aromatic white flowers.

Bacteria and fungi are able to grow in cosmetic products when nutrients are available and the conditions are satisfactory. For this purpose, preservatives should exhibit effective antimicrobial activity and be selectively toxic and effective against Gram-positive and / or Gram-negative bacteria, mold and yeast. However, due to the intrinsic characteristics, most preservatives of synthetic origin may present toxic reactions to the user, such as local irritation, contact dermatitis, hypersensitivity and others.

The proof of microbial quality in cosmetic products aims to prevent the transmission of diseases when they are used correctly, as well as ensuring the stability of the preparation and, consequently, its effectiveness. Cosmetics require preservatives capable of reducing the microbial load at acceptable levels during their use within the expiration date. Thus, it is necessary to evaluate the efficacy of preservatives in cosmetic formulations, determining the safe use of the product, aiming to test the conservative capacity of the cosmetic during its manufacture and during its shelf-life, throughout its use by the consumer.

In general, preservatives of synthetic origin have lipophilic characteristics, the use of aqueous or hydroalcoholic extracts may be more effective, since the growth of microorganisms occurs mainly in aqueous medium.

The use of active ingredients of natural origin in the production of cosmetics has grown a lot in the last years. The growing pursuit of effective and safe cosmetics has been constant.

The development of medicinal products and derivatives of vegetable origin is of great importance in the culture and tradition of human history. Most of the poor urban population, as well as small rural communities, trust and make great use of natural medicine, using plants as well as natural extracts to treat their illnesses. This is due to the cultivation of these populations and mainly due to the accessibility and availability of this type of treatment. As a consequence, there is a growing worldwide tendency to integrate traditional medicine and primary care (FENNEL, CW, LINDSEY, KL, MC. GAW, LJ, SPARG, SG, STAFFORD, GI, ELGORASHI, EE, GRACE, OM, VAN STADEN, J., Review: Assessing African medicinal plants forefficacy and safety: Pharmacological screening and toxicology (J. of Ethnopharmacol. 94, 205-217, 2004). Additionally, research into the development of new effective antimicrobial agents is necessary due to the development of microbial resistance and the occurrence of fatal opportunistic infections associated with AIDS, antineoplastic chemotherapy and transplants (PENNA, C, MARINO, S., VIVOT, E., CRUANES, M. C). , MUNOZ, J. DE D., CRUANES, J., FERRARO, G., GUTKIND, G., MARTI NO, V., Antimicrobial activity of Argentine plants used in the treatment of infectious diseases. (Ethnopharmacol. 77, 37-40, 2001). Numerous extensive research has been developed to find and classify antimicrobial agents from plant extracts and natural products, and a large number of plants represent a potential source in this search (DE SMET, PAGM, 1997, The role of plant - derived drugs and herbalmedicines. Healthcare Drugs, v. 54, no. 6, pp. 801-840; COWAN, MM, Plant products as antimicrobial agents Clinical Microb. Rev., v. 12, p.564-582, 1999; HERNÁNDEZ-PEREZ, M , RABANAL, RM, ARIAS, A., DE LA TORRE, MC, RODRIGUES, B. Aethiopinone, an antibacterial and cytotoxic agent from Salvia aethiopis roots Pharm. Biol. 37 (1), 17-21, 1999; SOKMEN, A JONES, BM, ERTURK, M., The in Vitroantibacterial activity of Turkiv medicinal plants J. of Ethnopharmacol.67, 79-86, 1999; KELMANSON, JE, JAGER, AK, VAN STADEN, J., Zulu medicinal plants with antibacterial activity J. of Ethnopharmacol.69, 241-246, 2000; SRINIVASAN, D., NATHAN, S., SURESH, T., PERUMALSAMY, O., Antimicrob ial activity of certain Indian medicinalplants used in folkloric medicine. J. of Ethnopharmacol. 74, 217-220,2001, MARTINI, N. D., KATERERE, D. R. P., ELLOF J. N., Biologicalactivity of five antimicrobial flavonoids from Combretum erythrophyllum (Combretaceae). J. of Ethnopharmacol. 93, 207-212, 2004; MITSCHER, L. A., DRAKE S., GALLAPUDI, S. R., OIUNTE, K., A modern look atfolkloric use of anti-infective agents. J. of Natural Product. 50, 1025-1040, 1987; CUTCHEON, A. R., STOKES, R. W., THORSON, L. M., ELLIS, S. M. HANCOCK, R. E. W., Anti-mycobacterial screening of British Columbian medicinal plants. Intem. J. of Pharmacognosy. 32.77-83. 1997; MENG, J.C., ZHU, Q.X., THAN, R.X., New antimicrobialmones and sesquiterpenes from Soroseris hookeriana subsp.Erysimoides, Medical Plant. 66, 541-544, 2000; RUBERTO, G., BARATTA, M. T., DEANS, S. G., DORMAN, H. J. D.,. Antioxidant andantimicrobial activity of Foenicullum vulgare and Criyhmum maritimumessential oils. Medical plant. 66, 687-693, 2000; HO, K. Y., TSAI, C.C., HUANG, U.S., CHEN, C.P., LIN, T.C, LIN, C. Antimicrobial activity of ethanol components from Vaccinium vitis-idaea, L. J. of Pharm. andPharmacol. 53, 187-191, 2001).

The frequently observed variation in results when plant extracts are tested for antimicrobial activity can be attributed to a number of factors. These include the assay technique, culture medium, bacterial or fungal strain, time, plant source (fresh or dry), and the amount of extract tested among others (FENNEL, CW, LINDSEY, KL, MC. GAW, LJ, SPARG , SG, STAFFORD, GI, ELGORASHI, EE, GRACE, OM, VAN STADEN, J., Review: Assessing African medicinal plants for efficacy and safety: Pharmacological screening and toxicology (J. of Ethnopharmacol. 94,205-217, 2004). However, these factors are no hindrance in the pursuit of standardization and further research involving the antimicrobial activity of natural extracts.

The study of the antimicrobial activity of substances used in pharmaceuticals and cosmetics is a subject that has been developing a lot, and only in the last 60 years has it been scientifically treated. Great importance is given to the subject, due to the concern not only with the microbiological aspect, but also with the potential of irritation and toxicity to the consumer (PINTO, TJA, KANEKO, TM, OHARA, MT, Biological quality control of pharmaceuticals, correlates and cosmetics. São Paulo: Atheneu, 2003).

The antimicrobial activity of plant extracts is evaluated by determining the smallest amount of the test substance needed to inhibit the growth of the test organism; this value is known as CMI (MADIGAN, MT; MARTINKO, JM; PARKER, J., Brock biology of Upper Saddle River, NJ: Prentice Hall, 2000. 9th ed., pp. 750-751). A very relevant aspect in determining the MIC of plant extracts is the concern, mentioned above, mainly regarding the toxicological, microbiological and legal aspects pertinent to natural compounds and / or combinations (PINTO, TJA, KANEKO, TM, OHARA, MT, Biological Quality Control). of pharmaceutical, related and cosmetic products São Paulo: Atheneu, 2003).

Research into new antimicrobial agents is necessary due to the emergence of resistant microorganisms and fatal opportunistic infections associated with AIDS, antineoplastic chemotherapy and transplants (PENNA, C, MARINO, S., VIVOT, E., CRUANES, M. C, MUNOZ, J. DE D., CRUANES, J., FERRARO, G., GUTKIND, G., MARTI NO, V., Antimicrobial activity of Argentine plants used in the treatment of infectious diseases.Insulation of activecompounds from Sebastiania brasiliensis J. of Ethnopharmacol. 77, 37-40, 2001). The study of antimicrobial agents has wide scope and is crucial in several sectors of the pharmaceutical and cosmetic field. Another point to be emphasized is that using the IMC study serves as the first screening to uncover the pharmacological activity of new agents.

Currently, there are several methods to evaluate the antibacterial and antifungal activity of plant extracts. The best known include diffusion in agar and macro and microdilution (PINTO, TJA, KANEKO, TM, OHARA, MT, Biological quality control of pharmaceutical, related and cosmetic products. São Paulo: Atheneu, 2003; COLLINS, CH, Antimicrobial susceptibility tests. In: _.

Microbiological methods. 7.ed. Oxford: Butterworth-Hunemann, 1995, p. 178-205; MADIGAN, M. T .; MARTINKO, J. M .; PARKER, J., Brockbiology of microorganisms. Upper Saddle River, NJ: Prentice Hall, 2000.9 ed. P. 750-751). In order to determine the MIC or Minimum Bactericidal Concentration (CMB) of active plant extracts, the sensitive microdilution method (ELOFF JN Asensitive and quick microplate method) has been widely used to determine the minimal concentration of plant extracts for bacteria. 64, pp. 711-713, 1998).

Stability may be defined as the ability of a product containing or not containing active substances to maintain their chemical, physical, microbiological, toxicological and bioactive properties within the limits specified previously during their shelf life, as these products when marketed pass through a storage system and may be subject to prolonged heat and humidity (ANVISA - National Agency for Sanitary Surveillance. Guide for Cosmetic Products Stability / National Agency for Sanitary Surveillance - Ia ed. - BrasíliaANVISA, 2004. 52 p. (Cosmetic Validity Series); vl) .

The study of the stability of cosmetic products provides information that indicates the degree of relative stability of a product under various conditions to which it may be subjected from its manufacture until its expiration. This stability is relative because it varies with time and as a function of factors that accelerate or delay changes in product parameters.

The microbial quality of a cosmetic and pharmaceutical product should be evaluated with the aim of ensuring efficacy and safety in its shelf life and condition of use. Verification of the microbial contamination levels of non-sterile products aims to prevent disease transmission and ensure product stability.

The presence of microorganisms above the acceptable limits may cause clinical problems to users and also, depending on the present organism, the toxic effect of the contaminated product endangers the patient's life (PINTO, TJA, KANEKO, TM, OHARA, MT, Biological Control of quality of pharmaceutical products, eco-correlates São Paulo: Atheneu, 2003). The relative danger created by the microbiological contamination of these products can be reported by the severity of the infection or disease caused. Microorganisms such as Pseudomonas, Proteus, Staphylococcus, Serratia, Streptococcus, Penicilium, Aspergillius and the Candida genus are classified as health hazards (BAIRD, RM, HODGES, NA, Denyer, SP. (Eds) (2000) Handbook of Microbiological Quality Control: Pharmaceuticals and Medical Devices Taylor & Francis, London, 2000, p.254).

One of the major sources of microbial contamination of cosmetic and pharmaceutical formulations is water (CROSHAW, B., Preservatives for cosmetics and toiletries. J. Soc. Cosmet. Chem., V.28, p.3-16, 1977; DIEHL, KH , Important secondary conditions for optimal use of cosmetic preservatives, Parfum Kosmet., Heidelberg, v.71, no.60, p.396-400, 1990; MALCOLM, SA, Microbiological monitoring and quality control., Manufacture Chem., Aerosol News, London, v.51, nov., p.55-56, 1980). The genus Pseudomonas is the most cited in the literature (ORTH, DS, Preservative Efficacy Testing of Aqueous Cosmetics and DrugsWithout Counting Colonies. Cosmet. & Toilet. 116: 41-47, 2001; TENEMBAUM, S. Considerations leading to the development of themicrobial limit guidelines of the CTFA Cosmetics & Toiletries, v.93, n.3, p.78-83, 1977; VAN ABBE, NJ HUGHES, O., DIXON, H.WOODROFFE, RS C, The Hygienic Manufacturing and Preservation of Cosmetics and Cosmetics J. Soc. Cosmet. Chem., 1970, v.21, p.719-800), but also found Xanthomonas, Flavobacterium, Achromobacter and Aerobacter. According to some authors; TENEMBAUM, S., Considerations leading to the development of the microbial limit guidelinesof the CTFA. Cosmet. Toilet, v. 93, no. 3, p. 78-83, 1977) cargabacterial reached the order of 10 6 CFU / mL.

Other important sources of microbial contamination refer to raw materials of natural origin (McCARTHY, TJ, Microbiological Control of Cosmetic Products. Cosmetics & Toilet, OakPark, v.95, n.8, pp. 23-27, 1980; 0 (NEILL, JJ, MEAD, CA, Congratulations: Bacterial Adaptation and Preservative Capacity (J. Soc. Chem. New York, v.33, p.75-84, 1982). The authors BONOMI and NEGRETTI have investigated the microbial quality of several of these raw materials, mainly of plant origin, usually employed in pharmaceutical preparations (BONOMI, E., NEGRETTI, F., Ricerche containing microbial content impiegatte nellepreparazioni farmaceutche. Ann. Ist. Super Sanita, v. 13, no. 4, pp. 805-832, 1977). Raw materials of synthetic or semi-synthetic origin generally have a low contaminating microbial load with the exception of some such as polyglycols or polyethylene glycols.

Products with their formulation and primary packaging contribute to microbial development because they can contain carbohydrates, fatty acids, proteins, amino acids, glycosides, fatty alcohols, steroids, peptides and vitamins. These substrates may serve as nutrients for contaminating microorganisms (MADDEN, I., JACKSON, GI, Cosmetic Preservation and Microbes: Viewpoint of the Food and Drug Administration. Cosmet. & Toilet, OakPark, v. 96, no. 10, p. 75- 78, 1981). Packaging material may be a source of contamination when not properly cleaned (SMART, R., SPOONER, DF, Microbiological spoilage in pharmaceuticals and cosmetics. J. Soc. Cosmet. Chem., V.23, 1972, p.721-737; VAN ABBE, NJ HUGHES, O., DIXON, H. WOODROFFE, RS C. The Hygienic Manufacture and Preservation of Toiletries and Cosmetics (J. Soc. Cosmet. Chem., 1970, v. 21, p.719-800). Components may interact with preservatives used in the formulation, such as parabens that can be adsorbed by plastic containers resulting in decreased preservative activity (BAHAL, SM & ROMANSKY, JM, Sorption of Congratulations byfexible tubings. Pharm Deu Technol., Aug; 6 (3) : 431-40, 2001). Another aspect to be considered is the contamination of the product by the user itself and appropriate recommendations on its use and the use of preservatives in the formulation are necessary.

Researchers BONOMI and NEGRETTI also investigated the microbial content of the raw materials employed in pharmaceutical preparations. According to these authors, the problems created by microbial contamination can be minimized by using raw materials that have no unacceptable bioburden history, adopting good manufacturing practices validated to reduce the risk of contamination during the process, and using them. effectiveness of preservative systems in formulations (BONOMI, E., NEGRETTI, F., Ricerche sul microbic content containing primimpiegatte nelle preparazioni pharmaceutche. Ann. Ist. Super. Sanita, v.13, n.4, p. 805-832, 1977).

Given all the factors that may contribute to contamination and aiming at product efficacy and consumer safety, the addition of preservatives in the formulations has been classically used. These are substances added to pharmaceutical and cosmetic forms in order to prevent microbial contamination. Its function is to inhibit the growth of microorganisms in the product, keeping it free from spoilage caused by bacteria, mold and yeast. They may have bacteriostatic and / or fungal activity, being used mainly in multi-dose products, which may present contamination. Choosing a suitable preservative system for a cosmetic product requires careful consideration of the product type, its use, deformation characteristics and likely microbial challenge. The definitive selection of preservative agent (s) should be a compromise between antimicrobial efficacy and product compatibility (PINTO, TJA, KANEKO, TM, OHARA, MT, Biological Quality Control of Pharmaceutical, Related and Cosmetic Products. Sao Paulo: Atheneu, 2003).

The effectiveness of the preservative system in pharmaceutical or cosmetic preparations is related to the composition of the formulation, the type of packaging material, the purity of the raw materials, the correct use by the consumer, the application form, and the preservative system itself. Even with all the strict manufacturing hygiene requirements (GMP), quality control and use of raw materials with permissible microorganism content, there is still the possibility of secondary contamination. However, some components, even if not typical antimicrobial substances, can help against the proliferation of microorganisms, increasing the effectiveness of preservatives. The general requirements for microbial growth are: water, energy, nitrogen sources, minerals and vitamins in an environment with adequate oxygen, pH and temperature levels (PINTO, TJA, KANEKO, TM, OHARA, MT, Biological Pharmaceutical Quality Control). , correlates and cosmetics São Paulo: Atheneu, 2003).

Preservatives, even those of natural origin, are extremely dry toxic substances, so the concentration used should be small. The preservative system used in a formulation may have a bacteriostatic effect, ie it maintains the same level of microorganisms or decreases, but does not destroy. Or even a bad effect of destruction. The action of the preservative system usually proceeds by combining the preservative chemical groups with the microbial proteins, or through the competitive action with certain enzymatic processes.

Description of the Figures

Figure 1 shows thin layer chromatography of L. salviaefolia ethyl acetate fractions.

Figure 2 refers to the spectra of fraction 4 of the ethyl deacetate fraction of L. salviaefolia. GC-MS analysis.

Figure 3 indicates the antimicrobial activity of L. salviaefolia total extract against S. aureus. Experiment performed in three replicates with three repetitions for each concentration. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control Cs: Solvent control; C +: Chloramphenicol (1 mg / mL), total extract solutions at different concentrations. Figure 4 shows the antimicrobial activity of the ethyl acetate fraction of L. salviaefolia in front of S. aureus. Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control; Cs: solvent control; C +: Chloramphenicol (1 mg / mL), solutions of the total acetate extract ethyl acetate fraction at different concentrations.

Figure 5 shows the antimicrobial activity of the chloroform fraction of L. salviaefolia against S. aureus. Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control; Cs: solvent control; C +: Chloramphenicol (1 mg / mL), solutions of the chloroform fraction of the extract in different concentrations.

Figure 6 refers to the antimicrobial activity of fractions (1-9) of the L. salviaefolia ethyl acetate fraction at a concentration of 1.0 mg / mL, compared to S. aureus. Experiment performed on three replicates with three replications. Values correspond to the mean of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Control degrowth; Cs: solvent control; C +: Chloramphenicol (1 mg / mL), solutions of ethyl acetate fractions (1-9) of total concentration at concentration of 1.0 mg / mL.

Figure 7 shows the antimicrobial activity of fraction 3 from ethyl acetate fraction against S. aureus. Experiment performed in three replicates with three replicates. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control Cs: Solvent control; C +: Chloramphenicol (1 mg / mL), fraction 3 solutions from the ethyl acetate fraction of the total extract at different concentrations

Figure 8 shows the antimicrobial activity of fraction 4 from the ethyl acetate fraction against S. aureus.

Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control Cs: Solvent control; C +: Chloramphenicol (1 mg / mL), fraction 4 solutions from the ethyl acetate fraction of the total extract at different concentrations.

Figure 9 shows the antimicrobial activity of L. salviaefolia extract against E. coli. Experiment performed in three replicates with three repetitions. Values correspond to the mean of each variable. Spectrophotometric determination at 630 nm. Variables = Cc:

Growth control; Cs: solvent control; C +: Chloramphenicol (1mg / mL), total extract solutions at different concentrations.

Figure 10 shows the antimicrobial activity of L. salviaefolia ethyl acetate fraction against E. coli. Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control; Cs: solvent control; C +: Chloramphenicol (1 mg / mL), solutions of the total acetate extract ethyl acetate fraction at different concentrations.

Figure 11 shows the antimicrobial activity of the decloroform fraction of L. salviaefolia extract against E. coli. Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control; Cs: solvent control; C +: Chloramphenicol (1 mg / mL), solutions of the chloroform fraction of the extract in different concentrations.

Figure 12 refers to the antimicrobial activity of fraction 3 from L. saluiaefolia ethyl acetate fraction against E.coli. Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control Cs: Solvent control; C +: Chloramphenicol (1 mg / mL), fraction 3 solutions from the ethyl acetate fraction of the total extract at different concentrations.

Figure 13 shows the antimicrobial activity of fraction 4 from L. salviaefolia ethyl acetate fraction against E.coli. Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables = Cc: Growth control Cs: Solvent control; C +: Chloramphenicol (1 mg / mL), fraction 4 solutions from the ethyl acetate fraction of the total extract at different concentrations.

Figure 14 shows the antimicrobial activity danaringenin, extracts and fractions of L. salviaefolia against S. aureus. Experiment performed in three replicates with three replicates. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables: Growth Control; Cs: solvent control; C +: Chloramphenicol (1 mg / mL); denaringenin solution at different concentrations.

Figure 15 demonstrates the danaringenin antimicrobial activity, extracts and fractions of L. salviaefolia against E. coli. Experiment performed in three replicates with three replicates. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables: Growth Control; Cs: solvent control; C +: Chloramphenicol (1 mg / mL); denaringenin solution at different concentrations.

Figure 16 shows the danaringenin antimicrobial activity, extracts and fractions of L. saluiaefolia against C. albicans. Experiment performed in three replicates with three replicates. Values correspond to the average of each variable. Photometric spectrum determination at 630 nm. Variables: Growth Control; Cs: solvent control; C +: Nystatin (1 mg / mL); naringenin solution at different concentrations.

Figure 17 shows the antimicrobial activity danaringenin, extracts and fractions of L. saluiaefolia against P. aeruginosa.

Experiment performed in three replicates with three repetitions. Values correspond to the average of each variable. Spectrophotometric determination at 630 nm. Variables: Growth Control; Cs: solvent control; C +: Amikacinal (1 mg / mL); naringenin solution at different concentrations.

Figure 18 shows the white 24h - Compostodermal-epidermal control after 24h in the air-liquid interphase culture system.

Figure 19 shows the dermal-epidermal compound after 24 hours of contact with the gel base without the addition of L. saluiaefolia.

Figure 20 refers to the dermal-epidermal compound after 24 hours of contact with the gel formulation incorporated with the L. salviaefolia ethyl deacetate fraction.

Figure 21 shows the dermal-epidermal compound after 24 hours of contact with the cream base without the addition of L. saluiaefolia. Figure 22 shows the dermal-epidermal compound after 24 hours of contact with the cream formulation incorporated with the L. saluiaefolia ethyl deacetate fraction. .

Figure 23 refers to the dermal-epidermal compound after 24 hours of contact with the shampoo base without the addition of L. salviaefolia.

Figure 24 shows the dermal-epidermal compound after 24 hours of contact with the formulation shampoo incorporated with the L. salviaefolia ethyl deacetate fraction.

Figure 25 shows the white 48hs - Dermal-epidermal control after 48hs in the air-liquid interphase culture system.

Figure 26 shows the dermal-epidermal compound after 48 hours of contact with the gel base without the addition of L. salviaefolia.

Figure 27 refers to the dermal-epidermal compound after 48 hours of contact with the incorporated gel formulation with L. salviaefolia ethyl deacetate fraction.

Figure 28 shows the dermal-epidermal compound after 48 hours of contact with the cream base without the addition of L. salviaefolia.

Figure 29 shows the dermal-epidermal compound after 48 hours of contact with the incorporated cream formulation of the L. salviaefolia ethyl acetate fraction.

Figure 30 shows the dermal-epidermal compound after 48 hours of contact with the shampoo base without the addition of L. salviaefolia.

Figure 31 shows the dermal-epidermal compound after 48 hours of contact with the shampoo formulation with incorporated with L. salviaefolia ethyl acetate fraction.

Figure 32 shows the cell viability through the study of human keratinocyte cytotoxicity of L. salviaefolia ethyl acetate fraction at different concentrations (0.0125%, 0.025%, 0.05%, 0.1%, and 0 , 2%) and different contact times (0.5h, lh, 2hs, 6hs, 24hs and 48hs).

Description of the Invention

The present invention relates to Lipia salviaefolia Cham. Leaf extracts, their active fractions, pharmaceutical and / or cosmetic compositions and their use as a natural antimicrobial (preservative).

In a first embodiment the present application is intended for Lippia salviaefolia Cham. Leaf extracts, such as hydroalcoholic extracts, dichloromethane, ethyl acetate and / or chloroform.

Preferably, the present application is intended for ethyl acetate and chloroform extracts of L. salviaefolia.

Active fractions are characterized in that they contain the active components, for example total flavonoids and total phenols, in particular, the active fractions contained in the fractions of the present invention refer to 4 ', 5-dihydroxy-7-methoxy-flavonone, naringenin and oapiflorol.

In a second embodiment, the patent application provides cosmetic and / or pharmaceutical formulations containing said extracts and / or their active fractions. In general, the formulations refer to emulsions, gels, creams, ointments, solutions, but not limited to these. In particular, the formulations of the present invention treat decrem, shampoos and gels. Cream compositions may be made with wax-emulsifier and / or surfactant, for example, polyglyceryl stearates, cetostearyl alcohol, PEG-20 methylglycoside sesquiestearate; optionally, polymeric consistency agents such as glyceryl omostearate, 2% w / v hydroxyethyl cellulose dispersion, such as emollient, mineral oil, cetyl acetate and / or lanolinacetylated alcohol, volatile silicone, 2-oethyl dodecanol; as a humectant, propylene glycol and / or glycerin; and preservatives such as parabens.

<table> table see original document page 20 </column> </row> <table>

The gel compositions may have in their composition polymeric and / or gelling agent now, for example, carbonyl polymers, hydroxycellulose; acidifying and / or alkaline agents such as 20% (w / v) sodium hydroxide solution, triethanolamine; surfactants such as cetostearyl alcohol, ethoxylated and / or propoxylated ethyl alcohol, fatty acid ester polyol as a wetting agent such as propylene glycol, sorbitol 70% and / or glycerin; preservatives such as parabens.

<table> table see original document page 21 </column> </row> <table>

The shampoo composition may contain in its formulation anionic, cationic and / or amphoteric surfactants and may be primary or secondary, such as sodium lauryl ether sulfate, disodium lauryl sulfate detriethanolamine, sodium lauryl sulfosuccinate and / or sodium lauryl sulfate diethanolamide. coconut, propyl betaine cocoamido, 3-oleyl phosphate; neutralizing agent such as 10% (w / v) citric acid solution; consistency agent such as glycol distearate, 20% (w / v) sodium chloride solution; and preservatives such as osparabens.

Composition Shampoo <table> table see original document page 22 </column> </row> <table>

In a third embodiment the present invention relates to the use of such extracts in the preparation of cosmetic and / or pharmaceutical formulations as antimicrobial and / or preservative. The antimicrobial activity of the fractions of Lippia salviaefolia Cham. Gram-negative parabacteria is indicated; Gram-positive yeasts and fungi (mold).

Still, the preservative system, resulting from the use of the fractionsative extracts of Lippia salviaefolia Cham. It has a broad spectrum of action, efficacy over a wide pH range, greater compatibility with other formulation components, minimal interference with organoleptic characteristics (such as color, odor, taste, etc.), being safe, nontoxic, non-irritating, nonsensitizing, adequate. oil-in-water partition coefficient to allow effective concentration in both phases, rapid inactivation of contaminants preventing microbial adaptation, easy handling, low cost, concentration stability.

Examples

For the fixation of the plant Lippia salviaefolia Cham, F.A.A. [37% (v / v) formaldehyde: glacial acetic acid: 70% (v / v) ethanol; (2: 1: 1)], stored in 70% (v / v) ethanol.

For histological description, free hand cuts were performed using a razor blade. The material was clarified in 70% (v / v) sodium hypochlorite solution and subjected to differential staining with 1% alcian blue (w / v) and 1% basic fuchsin (w / v). Leaf and transverse and transdermal cortical slices of limbus, petiole and stem were assembled in semi-permanent preparations, 50% (v / v) glycerin, with formalin drops and analyzed by Coleman binocular optical microscope.

To obtain the extracts, aqueous and hydroalcoholic, the leaves were previously desiccated in a greenhouse with a controlled temperature of 38 ° C and air circulation for four days, sequentially divided and sprayed in a knife mill with 1 mm diameter mesh.

The obtaining process was performed by extraction in Soxhlet for the hydroalcoholic extract; and decoction for the aqueous extract.

All solvents used in both extraction and chromatographic procedures were chromatographic grade. About 50 g of the electronic scale sprayed plant was weighed sequentially and extracted with PA ethanol until exhausted for three days at approximately 70 ° C. The dried extract was evaporated and subsequently lyophilized. With the residue from the previous extraction, a decoction extraction was performed from about 30 g of the plant. The extracts obtained were stored, properly packaged under refrigeration.

From the ethanolic and aqueous extracts, initial efficacy tests were made as natural preservatives.

The ethanolic extract was partitioned to verify the fraction with higher antimicrobial activity. 10.45 g of the lyophilized ethanolic extract which was dissolved in about 100 mL of hexane, weighed in an ultrasound bath, was weighed for 2 minutes and filtered under Büchner funnel under reduced pressure. The technique was repeated three times, thus obtaining the hexane fraction. In sequence, the residue from this first extraction, after filtration, was treated with chloroform using the same technique as the first fraction. After obtaining the chloroform fraction, the entire procedure was repeated using ethyl acetate as solvent to obtain the ethyl acetate fraction. Finally, the residue from the ethyl acetate fraction was extracted with n-butanol using the same technique, obtaining the butanol fraction. For drying of the extracts, a rotary vacuum evaporator was used.

Thus, four different fractions were obtained from the extract of L. salviaefolia. All fractions were concentrated by rotary evaporator and subsequently lyophilized.

For the fractionation of the ethyl acetate fraction obtained from the total extract of L. salviaefolia, the following chromatographic methods were used: Chromatographic Column and Thin Layer Chromatography (CCD).

An initial separation was initially performed through a column whose characteristics of the column used in the preliminary separation of the components of the ethyl acetate fraction (Table 1).

<table> table see original document page 25 </column> </row> <table>

1.0 g of the ethyl acetate fraction from the total extract of the leaves of Lippia salviaefolia was weighed, solubilized in methanol and incorporated into a small portion of silica (approximately 0.5 g), later deposited on the top of the column to perform separation. As a mobile phase, the dichloromethane: methanol mixture was used in different proportions as described in Table 2 which shows the mobile phase used in the column for the separation of the components of the L.salviaefolia ethyl acetate fraction and obtained fractions. All extractions were performed in duplicate, each fraction obtained was concentrated and lyophilized. After deliophilization, each fraction was submitted to the evaluation of its antimicrobial activity to identify the fraction with the best result.

Table 2

<table> table see original document page 25 </column> </row> <table>

The fractions obtained in the preliminary separation were again fractionated by thin layer chromatography. For this purpose plates containing a 1 mm layer of silica gel PF254 were employed. The eluent systems used were composed of dichloromethane and methanol in a volumetric ratio of 9: 1. Prior to the chromatographic run, the vat was previously saturated with the eluent system itself.

For the structural analysis, a gas chromatograph equipped with a flame ionization detector was used. The obtained spectra were compared with spectra banks of the apparatus itself as well as with literature spectral data.

In the study of antimicrobial activity, the hydroalcoholic and aqueous extracts, as well as the different fractions of the ethanolic extract, were diluted in dimethylsufoxide (DMSO): methanol (1: 1). Corresponding concentrations are expressed in terms of milligrams (mg) of the extract or fraction per milliliter (ml) of the solvent.

The antimicrobial activity of the extracts was evaluated against four types of microorganisms: Gram-positive bacteria:

Staphylococcus aureus (ATCC 6538); Gram-negative: Escherichia coli (ATCC 10536) and Pseudomonas aeruginosa (ATCC 9027); and fungus: Candida albicans (ATCC 10231).

As for the agar diffusion method, the tested organism was inoculated in a Trypic Soy Agar (TSA) slant for the bacteria, and a Sabouraud Dextrose Agar (SDA) for the fungus. After an incubation period of 24 h at 32 ° C for bacteria and 25 ° C for fungus, the cultures were suspended in sterile saline and adjusted to obtain a comparable turbidimetric reading as McFarland Standard No. 05. From these microbial suspensions, 1.0 mL was Dissolved in 4.0 mL of TSA for bacteria and SDA for fungus sequentially, these resulting 5 mL constituted the second layer of the Petri dish with a first 10 mL layer of microorganism agar. Aseptically, near the fire, appropriate tools were made some wells with approximately 6 mm in diameter, where the fractions and extracts were deposited to study their actions. In each Petriforam plate 5 wells were made: one used as a negative control, another positive for control, and the remaining three for triplicate samples.

Three concentrations were prepared for aqueous extract, and total ethanol extract: 20, 10 and 5 mg / mL. While two concentrations were prepared for the ethanolic extract fractions.40.0 µL of each solution (aqueous extract, ethanolic extract and fractions) were placed in the wells. In each Petri dish there was a well containing a 40 µl DMSO: Methanol (50:50) v / v solution used as a negative control, and as a positive control a well with 20 µl of a standard (1 mg / mL) chloramphenicol solution , (potency: 100,000UI / mL), for bacteria; nystatin (potency: 100,000 IU / mL) parafung; and amikacin (potency 916 µg / mg) for Pseudomonasaeruginosa. The plates were incubated for 24 h at 32 ° C (bacteria) and for 48 h at 25 ° C (fungus). The resulting inhibition zone diameters were measured by "Antibiotic Zone Reader - Fisher-Lilly" (mm). Each experiment was repeated at least three times. The measured zone of inhibition corresponds to the average diameter of the zone of inhibition found.

The microorganisms tested were: Staphylococcus aureusATCC 6538, Escherichia coli ATCC 10536, Candida albicans ATCC10231, Pseudomonas aeruginosa ATCC 9027, Aspergillus nigerATCC 16404. The evaluation of the antimicrobial activity of the extracts that showed better results in the agar and visually diffused C-agar test selection of extracts that have been incorporated into cosmetic formulations. The assay used was the microdilution method in liquid medium using aseptic techniques. For greater reliability of the results, the MIC determination of the selected extracts was evaluated after incubation with controlled temperature by microplate spectrophotometry. This test is an adaptation of the dilution procedure in liquid medium that uses larger volume (10 mL), with inoculum volumes, reducing sample and culture medium to 230 μL, allowing a larger number of replicates.

In an initial stage, the method was standardized and validated, the equipment used was the LM-LGC microplate reader providing precision spectrophotometric reading.

In each well were placed 200 (µL described below; C: negative control (medium); Cs: solvent control (medium, microorganism and solvent); Cc: growth control (medium and microorganism); C +: Positive control (medium , reference microorganism and antimicrobial);

Cext: extract control (medium and extract); and samples studied at appropriate concentrations. For all these variables, four wells are used for each microplate.

Through the study in gas chromatography-mass spectrophotometer (GC-MS) it was found that the three major-compounds whose fragmentation pattern, fraction 4 described in table 2, was compatible are 4 ', 5-Dihydroxy-7-methoxy-flavonone , naringenin, and apiflorol. These values served as a basis for comparison with the total extract and the active fractions of L.salviaefolia.

The cosmetic bases used in the present invention preferably involve emulsion (cream), gel and shampoo. Six test formulations were prepared (2 emulsions, 2 gels and 2 shampoos) selected from the sensory aspect by personal criterion (proper spread on skin, silky touch and aspect). These formulations were submitted to the preliminary physical stability tests (centrifugation and thermal stress), and to the accelerated stability test. At the end of these tests, an emulsion formulation, a gel and a shampoo formulation were chosen, with which the preservative efficacy test was continued. The compositions of each preliminary formulation are described in the following tables:

Table 3

<table> table see original document page 29 </column> </row> <table>

The procedure for preparing Cream A involved separately heating the aqueous and oily phases to a temperature of 70-75 ° C. When they reached this value, the phasase was poured over the oil slowly and steadily with the aid of mechanical agitation (Mixer). After obtaining the emulsion, it was homogenized until it was cooled to 40 ° C and the cyclomethicone, aessence and extract, previously diluted in q.s., were added. ethanol P.A. The depH value of the formulation was corrected to 6.0-6.5.

Table 4 <table> table see original document page 30 </column> </row> <table>

The aid of the plastic spatula was mixed with the Hostacerin® SAF and q.s. of flexible distilled water. The extract and extract were added and the system homogenized. The pH value of the formulation was corrected to 6.0-6.5.

Table 5

<table> table see original document page 30 </column> </row> <table> Hydroxyethylcellulose was hydrated with q.s. distilled water at 50 ° C and stirred with a flexible plastic spatula until gel formation. Propylene glycol, essence (previously dispersed in Procetyl® AWS) and extract were added and the gel was kneaded with q.s.p. distilled water and homogenized. The pH value of the formulation remained between 6.0-6.5.

Table 6

<table> table see original document page 31 </column> </row> <table>

The carboxyvinyl polymer was dispersed at q.s. distilled water and remained at rest for 3 h in order to complete hydration. After this period, propylene glycol was added and mixed with the aid of a flexible plastic spatula. The essence and Cetiol® HE were previously dispersed in Procetyl® AWSe added to the mixture, and then the extract was homogenized. The formulation was weighed, the mass was completed with distilled water and the pH was corrected to 6.0-6.5 with the aid of sodium hydroxide and manual homogenization.

<table> table see original document page 32 </column> </row> <table>

Sodium lauryl ether sulfate, triethanolamine lauryl sulfate and fatty acid diethanolamide were weighed and homogenized using a flexible plastic spatula.

50 g of distilled water were added with gentle stirring.

Dye, essence and extract were added, kneaded with distilled water and mixed gently. The pH was adjusted to 6.0-6.5 with citric acid, the sodium chloride solution was added until adequate thickening (visually observed and characteristic of a shampoo) and the system homogenized.

<table> table see original document page 33 </column> </row> <table>

Disodium lauryl sulfosuccinate, sodium lauryl sulfate (Genapol® SSB), sodium lauryl sulfate (Genapol® LSS), odiestearate glycol (Genapol® EGL), cocoamidopropyl betaine (Dehyton®KB) and phosphate 3-7 were weighed. (Hostaphat® 0-300) and gently homogenized with the aid of a flexible plastic spatula. They were incorporated into the formulation, q.s. of distilled water, the essence and the extract, the volume was made up with distilled water and mixed gently with the same stirring. The pH was adjusted to 6.0-6.5 with sodium hydroxide and the sodium chloride solution was added until adequate thickening (visually observed and characteristic of a shampoo) and the system homogenized.

All cosmetic preformulations were subjected to accelerated stability studies. Thus, in order to critically evaluate the preservative activity of L. salviaefolianas extracts pre-cosmetic formulations described above, they were subjected to drastic stress test conditions. Before starting the stability study the product was submitted to centrifugation and thermal stress test.

In centrifugation, ten grams of the formulations (cream A ecreme B) were rotated at 1000, 2500 and 3500rpm in centrifuge for fifteen minutes at each speed.

The samples (Cream A and B and Shampoo A and B) were placed in a thermostated water bath, between 40 and 80 ° C. Temperature elevation was performed every 10 ° C, maintaining each value for 30 minutes. Samples were evaluated at the end of the test (80 ° C), after returning to room temperature (22.0 ± 2.0 ° C).

After 24 hours of preparation, considered as ti (initial time), the chosen formulations, cream A and B, gel A and B and shampoo A eB, were subjected to the following conditions and evaluation periods:

On storage, the formulations were kept at room temperature (22.0 ± 2.0 ° C) for 14 days and evaluated on the 3rd, 7th and 14th days.

The product was exposed to sunlight indirectly and not directly to the sun. Because direct sunlight can significantly change the color and odor of the product and lead to degradation of the formulation.

The preparations were subjected to a temperature of 45.0 ± 2.0 ° C in a greenhouse for 14 days and evaluated on the 1st, 3rd, 7th and 14th days. In addition, the formulations were subjected to freezing cycles (-10.0 ± 2.0 ° C) for 24 hours in a freezer, followed by thawing and heating in a greenhouse (50.0 + 2.0 ° C) for 24 hours, completing -this form a cycle. The samples were submitted to six cycles, being evaluated on the 3rd (6th day) and 6th cycles (12th day).

Also, the formulations were kept at a temperature of -10.0 ± 2.0 ° C at. freezer for 14 days and evaluated on the 7th and 14th days. The evaluation was still in a refrigerator where the formulations were kept at (6.0 ± 2.0 ° C) for 14 days and evaluated on the 3rd, 7th and 14th days.

After 24 hours of preparation (ti), each formulation was evaluated for organoleptic characteristics (appearance, color and odor), depH value and apparent viscosity. The results obtained in ti were considered as reference for comparison with the other sample readings, under all conditions, during the Accelerated Stability Test.

Challenging microorganisms used in the validation and efficacy of preservatives were: Escherichia coli (ATCC 10536), Pseudomonas aeruginosa (ATCC 9027), Staphylococcus aureus (ATCC6548), Burkholderia cepacia IAL 1834 ATCC (17759), Candida albicans (ATCC 10231) and Aspergillus niger (ATCC 16404).

The choice of the preservative system inactivation condition aims to validate the method used in the preservative system efficacy test. The reliability of the method used in the preservative efficiency test depends on its ability to prevent preservative system interference with the growth of microorganisms.

For the choice of neutralization or inactivation condition of the preservative system, the study was conducted with formulations of A, Gel B and Shampoo B. These products were prepared in aseptic conditions and considered as the test sample. To control the viability of microorganisms, 0.9% (w / v) saline was used.

The samples of Cream A, Gel B and Shampoo B were evaluated for preservative system efficacy. The challenge test was performed for each of the above mentioned microorganisms.

Approximately 20g of each test sample was inoculated with one of the challenging microorganisms at about 10 5 to 10 6 CFU / g from the previously standardized suspension as described above and respecting the ratio of inoculum volume to suspension to be less than or equal to 1. % (w / v) of the sample volume.

Immediately after inoculation, it was homogenized for one minute on a shaker and subjected to serial dilution and counting of viable microorganism colonies (CFU / g) by the deep sowing technique. For the decimal dilution procedure, the Dey Engley (D / E) odiluent was used. As culture medium, soycasein agar and sabouraud-dextrose 4% agar were used.

The challenged samples and controls were kept at room temperature throughout the test. The periodicity of each preservative system evaluation of the samples was maintained. It is noteworthy that, for all methods used to evaluate the efficacy of the preservative system, tests were performed immediately after inoculation of the microorganism, considered time zero (to).

For the verification of the viability of the microorganisms during the tests, the same procedure used for the sample was performed, although employing 0.9% (w / v) saline, considered in the test as control of the viability of the microorganism. promptly after inoculation and after 2, 7, 14, 21 and 28 days. They were named respectively to, t2, t7; ti4; t2i and t28-

For the safety evaluation of the cosmetic formulations and incorporation of L. salviaefolia in histological studies, dermal-epidermal skin substitutes were initially prepared from the dermal bases. Subsequently, secondary keratinocyte cultures were prepared on the dermal bases and cultured at the air-liquid interface. The dermis was prepared with 2x2 cm2 and kept in PBS (Phosphate Buffered Saline), a ring was placed over the dermis to seed high density keratinocytes, leaving overnight to remove the ring. On the seventh day the set was passed to the six-well plate network system. The cultures were kept in a greenhouse with 5% CO2 at a constant temperature of 37 ° C, and in each well contained 3 mL of KGM (Keratinocyte GrowthMedium), the culture medium was changed every two days until 14 ° C. Day and daily until the 21st day. To begin the safety evaluation test, each medium was aspirated and, with the aid of a 1mL insulin syringe, 1 drop (about 20-30 nL) of each sample (gel, cream exampu without and with the incorporation salviaefolia) on compostcultivated in the air-liquid interphase, in a single replicate, maintaining a well as control without the addition of a sample.

After being added to the samples in the compounds grown on air-liquid interphase, the systems were maintained for 24 hours or 48 hours in the culture medium. The dermo-epidermal system was washed 3 times with PBS and transferred with tweezers and needle to a filter paper. The system was sequentially treated and fixed by optical paramicroscopy for 24-72 hours in a refrigerator, comparing 4% formaformaldehyde (PFA) in sodium cacodylate buffer pH 7.2. It was foiled again three times with PBS and left in 70% alcohol in the refrigerator. The analysis of the morphology of the human skin, dermo-epidermal system, was made under optical microscope, with magnification of 1000 times (Labophot - Nikon microscope).

A stock solution of 2 mg / mL was prepared by solubilizing the dry extract of L. salviaefolia empolyethylene glycol 400 (PEG 400) ethyl acetate. The low-density (with support layer) HK186 keratinocytes formed by 60 Gy previously irradiated CC1-92 fibroblasts in the P0 passage were amplified into a 75 cm 2 bottle in KGM medium. From primary deculture, after reaching the required amount of cells, they were trypsinized and seeded at 50,000 cells / well of HK186 in two 96-well multiwell plates. After 24 hours 100 µl of the necessary serial dilutions of the L.salviaefolia stock solution in PEG were added to reach concentrations of 2%, 1%, 0.5%, 0.25% and 0.125% (w / v) of the fraction. of ethyl acetate from L. salviaefolia extract in freshly prepared and previously sterilized filtration sterile KGM medium in triplicates.

The samples were kept in contact with this L. salviaefolia solution for 2, 6, 24 and 48 hours. A treatment control was also made where the KGM culture medium was added in 11 replicates and incubated until the end of the test (48 hours). And the assessment of solvent interference: after the incubation period the culture medium was exchanged by KGM, containing increasing amounts of solvent 0%, 5%, 1%, 2%, to be tested and then plated again incubated at time intervals. At 2, 6, 24 and 48 hours, for the time interval of 2 hours only the concentrations of 2% and 1% were used, ie, reproducing the most drastic conditions of the cell viability test for L. sage. After these pre-established contact periods, the culture medium was exchanged for KGM and the samples were kept until the end of the test, 2 days, and then subjected to rhodamine staining (qualitative assessment) and eMTS (quantitative assessment) to verify cell viability. .

After 48 hours the solutions contained in the wells were unspirated and 120 aliquots of the MTS / PMS solution in KGM were added to all wells of the plate, which was immediately conditioned in a controlled atmosphere oven at 37 ° C with 5% CO2 and 95% relative humidity for 2 hours. After this initial period, the first absorbance measurement at 490 nm was performed on a printer-coupled ELISA plate spectrophotometer. The plate was incubated again and three further readings were taken at 1 hour interval.

In order to quantify cell proliferation, the colorimetric method entitled Cell Titer 96® was applied.

For preparation of the MTS / PMS solutions in KGM: MTS and PMS solutions were thawed in a thermostated bath at 37 ° C for 10 minutes. 15.873% MTS was transferred to a conical bottom tube containing 83.333% cell culture medium (KGM). Then 0.794% PMS was added while shaking the solution vigorously.

The hydroalcoholic and aqueous extracts were obtained after finding the inefficiency of the aqueous extract as an antimicrobial agent, and the action of the hydroalcoholic extract was made to partition the hydroalcoholic extract to obtain the fractions and study their antimicrobial activity in order to identify the fraction with the best antimicrobial activity. Tables 9 and 10 describe the dry weight and yield of each extract and fractions. Table 9 refers to the yield of aqueous and hydroalcoholic extracts obtained from L. salviaefolia.

Table 9

Yield of aqueous and hydroalcoholic extracts of L. salviaefolia

<table> table see original document page 40 </column> </row> <table>

Table 10 shows the yield of fractions obtained from the partition of the L. salviaefolia hydroalcoholic extract.

Table 10

Yield of fractions of L. salviaefolia hydroalcoholic extract

<table> table see original document page 40 </column> </row> <table>

The fraction with the highest yield is the ethyl deacetate fraction, with a yield of 41.34% (Table 10). After partitioning and study of antimicrobial activity by diffusion of the hexanic, chloroform, and ebutanolic ethyl acetate fractions. The most effective active fractions were found to be: ethyl acetate fraction and chloroform fraction. After a CCD analysis, it was found that the chloroform and ethyl acetate fractions had similar composition. Due to the similar composition and IMC of the two fractions are similar, the ethyl acetate fraction has a higher mass, better yield and easier handling due to the lower concentration of fatty compounds.

From the chromatographic column extraction of the ethyl deacetate fraction we obtained fractions 1, 2, 3, 4, 5, 6, 7, 8 and 9. Each of these fractions was submitted to the study of antimicrobial activity at 1.0 mg / mL front. S. aureus, microorganism more sensitive to antimicrobial activity tests. Enabling the identification of fractions with considerable antimicrobial activity and consequently of greater interest for the study in question. Fractions 3 and 4 presented better antimicrobial activity.

The analysis in CCD was performed as a "screening", allowing identification of the fractions that present the most common compounds. The major compounds were found to be in fractions 3 and 4. As well as the best antimicrobial activities. Constitutional analysis was performed by the CG-MS.

The structural analysis was performed only for the most purified fractions with higher antimicrobial activity, because in these fractions there are components of interest, with greater microbial activity. Due to the similarity of composition between fractions 3 and 4, observed in CCD, only fraction 4 was submitted to structural analysis by GC-MS. GC-MS analysis of fraction 4 obtained with the separation procedures revealed the presence of flavonones. The three major major compounds whose fragmentation pattern is compatible are 4 ', 5-Dihydroxy-7-methoxy-flavonone (retention time = 17.032), naringenin (retention time = 17.537 min.), And apiflorol (retention time = 17,988).

An initial screening was used to check fractions and extracts with antimicrobial activity. The results of antimicrobial activity by the agar diffusion method of ethanolic aqueous extracts and ethanolic extract fractions are present in natabela 11. The ethanolic extract showed good antimicrobial activity against S. aureus, E. coli, and C. albicans. The measured inhibition area diameters, referring to the studied extracts were between 13.3 -1.3 mm depending on the studied microorganism.

Table 11 shows the antimicrobial activity measured by the growth inhibition diameter of L. salviaefoliae fractions extracts by the agar diffusion method. Where i: inactive; -: unrealized test; EE: ethanolic extract of L. salviaefolia, EAq: aqueous extract of L. salviaefolia, Fr.H .: hexane fraction; Fr.CHCb: chloroform fraction; Fr.Ac.Et .: ethyl acetate fraction; Fr.ButOH .: butanolic fraction.Table 11

Agar method and diffusion, inhibition halo diameter (mm) of L. salviaefolia extracts and fractions

<table> table see original document page 43 </column> </row> <table>

The hexanic and butanolic fractions showed little activity against the tested microorganisms. However, the use of chloroform and ethyl acetate fractions were effective, which corroborates a good antimicrobial activity against S. aureus, E. coli eC. albicans, among others.

According to the results presented in table 11, it can be noted that the values for the inhibition zones for ethyl acetate fraction are very close to those presented by the chloroform fraction, and the values of the inhibition zones for ethyl acetate fraction are slightly lower than the values corresponding to the chloroform fraction.

The antimicrobial activity of aqueous extract, total ethanol extract and fractions of L. salviaefolia was tested by the agar diffusion method against four microorganisms: one Gram-positive (S.aureus), two Gram-negative {E. coli and P. aeruginosa) and a fungus (C.albicans). The results showed considerable antimicrobial activity for the total ethanolic extract and for the chloroform and ethyl acetate fractions.

The extracts of L. salviaefolia shown here report to be a functional active in dosages lower than those presented in the state of the art regarding the diffusion method. Therefore the agar diffusion method was used only as a screening to find the active fractions. The MIC of the total extract as two fractions of L. salviaefolia was determined by the micro-dilution method. The results regarding the MIC determination confirmed the results presented by the agar diffusion test. Hexane and butanol fractions are inactive. However, as can be seen in natabela 8, the total extract, chloroform fraction, ethyl acetate fraction, and fractions 3 and 4 from ethyl acetate fraction were active in front of S. aureus, E. coli, C. albicans and A. niger. . On the other hand, only chloroform and ethyl acetate fractions were active against P.aeruginosa.

In Table 12 the minimum inhibitory concentrations (MIC) for paranaringenin are presented for the total extract of L. salviaefolia, for the active fractions of the total extract: ethyl acetate fraction and chloroform fraction; and for fractions 3 and 4 derived from the ethyl deacetate fraction. As it can be observed, fractions 3 and 4 derived from ethyl acetate fraction did not present higher MIC than those of their original fraction, making the purification of ethyl acetate and chloroform fractions unjustifiable, this factor can be explained by the favorable effects between substances. present in the extracts.

Table 12 shows the MIC of naringenin, L. salviaefolia total extract and fractions by the microdilution method, against abacteria and fungi, being EE: ethanolic extract; CHCb.Fr: Chloroform fraction of the ethanolic extract; Ac.Et.Fr .: Fraction of ethyl acetate ethanolic extract; Fr. 3: Fraction 3 of the ethyl acetate fraction; Fr. 4: Fraction 4 of the ethyl acetate fraction.

Table 12 Minimum Inhibitory Concentration of L. salviaefolia (IMC mg / mL) Microdilution Method

<table> table see original document page 46 </column> </row> <table>

According to Table 12, it was observed that when performing the L. salviaefolia antimicrobial activity test, the chloroform fraction and the ethyl acetate fraction had the highest antimicrobial activity, in which they presented a 0.2% MIC for asbacteria and Fungi tested.

Fractions 3 and 4 from the ethyl acetate fraction presented similar MICs when compared to each other (Table 12).

This fact can be explained due to the constitutional similarity between fractions. However, the MIC presented by fraction 3 and fraction 4 were not more effective than the ethyl acetate or chloroform fraction (table 12). This fact can be explained due to the synergistic effect presented by the constituents of each fraction. Thus, for L. salviaefolia, as the fraction was purified it decreased antimicrobial activity. Three major compounds were isolated in GC-MS. The major major compounds whose fragmentation pattern is compatible are 4 ', 5-Dihydroxy-7-methoxy-flavonone, naringenin, and apiflorol. Among these compounds naringenin is found for commercialization. In this way the antimicrobial activity for naringenin was studied. Naringenin as well as the total extract of L. salviaefolia (Table 12) was found to have the highest MIC values found in the study of antimicrobial activity. For S.aureus the CMI of naringenin and total extract were between 0.125-0.25mg / mL; for E. coli, MIC between 1.0 - 1.5 mg / mL; for P. aeruginosa> 2.0 mg / mL; for C. albicans between 1.5 - 2.0 mg / mL; and for A. niger between 1.5 - 2.0 mg / mL. This fact shows that in order to reach the best levels of L. salviaefolia antimicrobial activity, the initial extraction process and the total extract partition must be done until the chloroform fraction and the ethyl acetate fraction can be obtained. From this point on if purification and isolation of the compounds proceed to antimicrobial activity try to decrease.

The high efficiency of the ethyl acetate fraction and the chloroform fraction can be explained by the synergistic effect between all components of these L. salviaefolia fractions. This fact can be explained due to the constitutional similarity of the two fractions observed in the CCD test.

The reason for the difference between the sensitivity of Gram-positive and Gram-negative bacteria is due to the difference in the constitution of each cell wall. Gram-negative bacteria have a phospholipid membrane carried with lipopolysaccharide components. This makes the cell wall impervious to some antimicrobial chemicals. On the other hand, Gram-positive bacteria are more susceptible since they have a cell membrane consisting only of a depeptidioglican layer and are not an effective permeability barrier.

However, in relation to these permeability differences, L.salviaefolia was effective for both Gram-positive and Gram-negative, especially the fractions: echloroform ethyl acetate (Table 12).

A. niger is usually insensitive to most plant extracts tested for antimicrobial activity, and C. albicans was a difficult to inhibit fungus. However, as can be seen in table 12, both the chloroform fraction and the L. salviaefolia ethyl acetate fraction were active with considerable MIC values. Also, all tested extracts of L. sage and folia had activity against bacteria and fungi, especially the chloroform fraction and the ethyl acetate fraction. Indicating good antimicrobial activity.

After centrifugation of emulsions: cream A (CA) and cream B (CB), no coalescence or phase separation was observed, demonstrating adequate physical stability.

The formulations of creams A and B remained stable, withstanding the temperature changes to which they were subjected in this heat stress, showing no phase separation.

Results on organoleptic characteristics, pH values and apparent viscosity of the formulations initially evaluated and after exposure to the test conditions (4-8 ° C refrigerator, 45 ° C oven; freeze-thaw cycles; -10 ° C freezer; ambient temperature at 22 ° C and sunlight) for creams A and B, gels A and B and shampoos A and B.

The presence of microorganisms at certain levels and types in non-sterile products may be a condition related to product deterioration, as well as non-compliance with Good Manufacturing Practices may also lead to production with inadequate sanitary quality. This process can even be observed in cases where the substrate offers minimum conditions necessary for microbial development. Surviving contaminants multiply in poor nutrient environments or adapt to very unfavorable conditions. The proliferation of microorganisms can cause changes of various kinds, such as loss of the active substance content and physicochemical and / or organoleptic changes of the product.

For cosmetics, a limit level of viable organisms is allowed, the assay consists of purposeful contamination of the sample with standard organisms and the survivors' load is determined for 28 days using a validated microbial counting technique.

Comparing to the conditions studied, it is observed that only under condition 2 was it possible to recover all microorganisms (Table 13). These results indicated that for the enumeration of microorganisms there would be a need for a 10 "dilution of the Cream A, Gel B and Shampoo B formulations with Dey Engley Diluent (D / E). Based on these results for the conservative efficacy test, we proceeded to dilution of the formulations prior to enumeration or counting of viable microorganisms in the periods immediately after inoculation, after 2, 7, 14, 21 and 28 days.

Table 13 presents the results of microorganism recovery under the two neutralization conditions, where the control = sterile 0.9% (w / v) saline; test sample = product prepared with preservative under aseptic conditions and each digit represents the average of the test performed on five replicates. It is observed that the control presented the recovery always greater than 70% for all microorganisms, demonstrating the viability of the microorganisms during validation and ensuring that the recoveries smaller than 70% were due to the inadequate condition for the microorganisms growth.

Table 13

<table> table see original document page 50 </column> </row> <table>

To assess the efficiency of the preservative system, the test consists of contamination of Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, Candida albicans and Aspergillus niger in each sample and the survival rate of the survivors determined at pre-established times for 28 days. validated conventional microbial counting technique.

The results of the preservative activity evaluation, 0.2% (w / v) extract, in Cream A, Gel B and Shampoo B formulations are presented below.

Table 14 shows the burden of Escherichia coli ATCC 10536 survivors (CFU / g or mL) on the preservative efficacy test, where to = count immediately after inoculation; t48 = count at 48 hours after inoculation and t7, ti4, t2i and t28 = count at 7, 14, 21 and 28 days after inoculation.

Table 14

<table> table see original document page 51 </column> </row> <table>

Table 15 shows the survivor burden of Pseudomonas aeruginosa ATCC 9027 (CFU / g or mL) on the preservative efficacy test, where t = count immediately after inoculation, U8 = 48 hour count after inoculation and t7, ti4, t2i and tas = count at 7, 14, 21 and 28 days after inoculation.

Table 15 <table> table see original document page 52 </column> </row> <table>

Table 16 shows the survivor burden of Staphylococcus aureus ATCC 6538 (CFU / g or mL) on the Preservative Efficacy Test, where t = count immediately after inoculation, t48 = 48 hour count after inoculation and t7, ti4, t2i and t28 = count at 7, 14, 21 and 28 days after inoculation.

Table 16

<table> table see original document page 52 </column> </row> <table>

Table 17 shows the Burkholderia cepacia ATCC survivor burden (CFU / g or mL) in the Preservatives Efficacy Test, where t = count immediately after inoculation; Us = count at 48 hours after inoculation and t7, ti4, t2i and t28 = count at 7, 14, 21 and 28 days after inoculation.

Table 17

<table> table see original document page 53 </column> </row> <table>

Table 18 shows the Candida albicans ATCC 10231 survivor load (CFU / g or mL) in the Preservatives Effectiveness Test, where t = count immediately after inoculation; t48 = counting at 48 hours after inoculation and t7, ti4, t2i and t28 = counting at 7, 14, 21 and 28 days after inoculation.

Table 18

<table> table see original document page 53 </column> </row> <table> Table 19 refers to the AspergillusnigerATCC 16404 survivor load (CFU / g or mL) in the Preservative Effectiveness Test, where to = counting immediately after inoculation; Us = count at 48 hours after inoculation and Δ7, ti4, t2i and t28 = count at 7, 14, 21 and 28 days after inoculation.

Table 19

<table> table see original document page 54 </column> </row> <table>

The tests were performed, concomitantly with 0.9% (w / v) saline solution inoculated with the same microbial load of the test sample, the suspension was considered as control to verify the viability of the challenging microorganisms during the 28 days of the test. In this condition, it was found that for Escherichia coli (Table 14), Pseudomonas aeruginosa (Table 15), Staphylococcus aureus (Table 16) and Burkholderia cepacia (Table 17) bacteria were viable during all tests, as well as for Candida fungi. Albicans and Aspergillus niger which also remained adequate over the evaluation time.

According to the analytical results described in nastables 14, 15, 16, 17, 18 and 19, it can be stated that: the samples decememe, gel and shampoo in the concentration of 0.2% of the ethyl acetate extract fraction of L. ethanolic extract. Salviaefolia meet specification adopted for bacteria: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Burkholderia cepacia, and Candida albicans.

After the contact period of 24 hours or 48 hours, samples with the dermal-epidermal skin substitute, hematoxylin-eosin staining and cut, the effect of the bases without incorporation of L. salviaefolia and of the cosmetic formulations with the incorporation of L. salviaefolia was observed. , in the epidermis of each dermal-epidermal system. Thus assessing the safety of each formulation.

In Figure 18 we have the control called white 24h in which no sample was added, but remained in the air-liquid interphase culture system during the 24 hours of testing. In this illustration, the epithelium was organized and reproducing the expected morphology for the dermis and epidermis, with dye differences due to cell maturation, and the keratin lamellae can be observed, constituting the corneal layer. In addition to the observation of intermediate or thorny and granular layers.

Figure 19 shows the histological study of the skin after 24 h contact with the gel-based formulation, observed under microscopy, reveals the morphology of the epidermis and dermis, although slight dermo-epidermal separation may be observed due to the slight change in the morphology of the epidermis. epidermis.

Figure 20 shows the histological study of the skin after 24 hours contact with the cosmetic gel formulation, incorporated with L. salviaefolia as a natural preservative. In this case a higher toxicity of the formulation can be observed since there was a significant alteration in the morphology of the epidermis, and death of most keratinocytes. Found due to the impossibility of identifying these cells as can still be seen in figure 18. This higher toxicity when comparing the gel without the incorporation of L.salviaefolia is due to the inherent toxicity of the extract, but it is important to note that the formulation itself without the addition of L Salviaefolia had already shown some toxicity.

The cream formulation had the lowest toxicity while retaining dermis and epidermis with normal morphological characteristics after contact time of 24 h (Figure 21).

Figure 22 shows the influence of the cream cosmetic formulation incorporated with L. salviaefolia, although the lower keratin levels showed a low toxicity.

Also observing viable keratinocyte cells in the epidermis.

In addition to maintaining the morphology of the dermis and epidermis.

Figure 23 shows the toxicity of shampoo formulation even without the addition of L. salviaefolia. This is due to the presence of surfactants, so they are mostly used in rinse-off and external products. Figure 23 shows the detachment of the entire keratin corneal part of the epidermis and its own structural alteration of the epidermis mainly in the middle layer, although it is still possible to observe viable keratinocyte cells mainly close to the dermal papillae in the basal layer.

Figure 24 shows the dermal-epidermal compound after 24 hours of contact with the shampoo cosmetic formulation incorporated with L.salviaefolia. The toxic effects are similar to the effects shown in Figure 23 representing the shampoo base without the addition of the ethyl acetate fraction of L. salviaefolia extract. In which there is a detachment of the corneal layer and the dermo-epidermal separation, with structural alteration of the epidermis. Characteristics evidenced by shampoo base toxicity and L. salviaefolia toxicity, however, all the toxicity found in figures 23 and 24 should not be attributed only to the extract since the formulation without the extract was also toxic, probably due to the presence of additives in the formulation.

In Figure 25 we have the control called blank 48 h and no sample was added but remained in the air-liquid interphase culture system during the 48 hours of testing. In this illustration the epithelium was organized despite the separation of the dermis and the epidermis, and in the evaluation of the toxicity for this can be an artifact, thus the toxicity was evaluated according to the aspect of the morphological differences and tintorial differences of the skin. Figure 25 shows tintoral differences due to cell maturation, and can also be observed the keratin lamellae, constituting the corneal layer. In addition to the observation of intermediate or thorny and granular layers.

Figure 26 shows the influence of the base gel sample on 48 hours of contact with the dermal-epidermal compound. In this illustration we only see the epidermal part where it is possible to verify the toxicity of the gel base due to the morphological and dye characteristics present in the epidermis, mainly in the visualization of keratinocyte cells that are not evident and the detachment of the cornea.

Figure 27 shows the toxicity of the L.salviaefolia gel in contact with the dermal-epidermal compound, noting differences in morphology and tinting aspects, detachment of the dermis and epidermis, detachment of the superficial layer of the cornea, difficulty in identifying viable keratinocytes. However, it is important to note that in this figure we have the toxicity of the base formulation associated with the toxicity of L. salviaefolia. And when compared to figure 26 there are no significant differences. This suggests that some of the toxicity is due to the constituents of the base formulation itself.

The histological study by optical microscopy aiming to evaluate the toxicity of the cream cosmetic formulations for 48 h of contact is shown in figures 28 and 29.

Figure 28 shows only the influence of the base of the formulation without the addition of L. salviaefolia and Figure 57 has the formulation incorporated with the plant extract. In both figures we only see the epidermis. In figure 28 you can more easily visualize viable keratinocytes.

While in Figure 29, the presence of viable keratinocyte cells is not as easily detectable, suggesting greater toxicity presented by the formulation with the incorporation of L.salviaefolia.

The toxicity evaluation of the base exampu shampoo formulations incorporated with L. salviaefolia in dermal-epidermal compound are elucidated in figures 30 and 31. Significant setoxities were found in both formulations due to morphological and dye differences in the epidermis.

Figure 30 shows the separation of the dermis and the epidermis in addition to the separation of the horny layer and the superficial layer in the epidermis, in addition to the difficulty in showing viable keratinocyte cells.

Similar to Figure 30, Figure 31 shows the separation of the dermis and epidermis; however, the epidermis did not present major morphological alterations, which characterizes a similar detoxicity level between the formulations with and without the incorporation of L. salviaefolia. Base formulations, without the incorporation of L. salviaefolia, it was found that even the bases of the formulations, especially in the shampoo formulation, exert a toxic effect on the cultures of dermal-epidermal skin substitutes. Thus in some cases it cannot be stated that such toxic effects are due solely to the Lippia saviaefolia ethyl acetate fraction.

In the cytotoxicity experiment in human keratinocytes culture the L. salviaefolia ethyl acetate fraction was placed directly on the keratinocytes. Thus the test is more sensitive than the in vivo test or even, thus the results are more safely exposed in relation to dermal-epidermal toxicity studies. Also highlighting the relationship of various concentrations and different contact time of the ethyl acetate fraction of L. salviaefolia in contact with human keratinocytes.

According to the results obtained in the cytotoxicity test (Figure 32), it was found that L. salviaefolia at a concentration of 0.2%, corresponding to the MIC, was toxic in almost all contact times, except for half an hour of contact, where a cell viability of about 90% is observed.

At the concentration of 0.1% of the ethyl acetate fraction of L.salviaefolia, cytotoxicity was observed at 24 and 48 hours.

However at this same concentration (0.1%) with 6 hours of contact achieved a cell viability of 70%, reaching safety at 2 and half hours of contact (Figure 32).

At a concentration of 0.05% of the ethyl acetate fraction daL. salviaefolia, with 48 hours of contact demonstrated a cell viability of about 70%. At this same concentration with 24 and 6 hours of contact, cell viability was around 80%, reaching the safety range with half and 2 hours of contact (Figure 32).

Already at concentrations of 0.025 and 0.0125% of ethyl acetate of sage sage, only 48 hours of contact observed levetoxicity in which cell viability was between 80 and 90%. In the other tested contact times, they were safe (Figure 32), suggesting that cosmetic formulations for external use, containing up to 0.025% of L. salviaefolia ethyl acetate, may be considered safe.

Thus, it is suggested that the L.salviaefolia ethyl acetate fraction in the MIC concentration (2.0 mg / mL) showed serocytotoxicity only after 6 hours of contact, and with 2 hours of contact reached a cell viability of. just under 30%, reaching safety and cell viability with only half an hour of contact with profile similar to the control.

The present application presents an absorption assessment as a perspective to ascertain which L.salviaefolia concentration actually comes into contact with viable keratinocytes.

However, even with the toxicity determined, L. sage can be used as a preservative in rinse formulations, such as shampoo, where legislation permits the use of more aggressive substances. Another possible use for L. salviaefolia is intended as a selective antimicrobial. According to the present invention, the MIC for the ethyl acetate fraction has values within the safety limit for S. aureus, with MIC of 0.035 - 0.005 mg / mL; for C.albicans with an MIC of 0.25-0.375 mg / mL and for E. coli with an MIC of 0.5-0.75 mg / mL.

Claims (14)

1. LIPPIA SALVIAEFOLIACHAM. LEAF EXTRACTS, characterized in that it comprises hydroalcoholic extracts, dichloromethane, ethyl acetate and / or chloroform. Extracts according to Claim 1, characterized in that the extracts preferably comprise ethyl acetate and / or chloroform extracts. Extracts according to Claims 1 and 2, characterized in that the extracts comprise active fractions. Active fractions according to claims 1 to 3, characterized in that the active fractions comprise the actives chosen between total flavonoids and total phenols, in particular 4 ', 5-dihydroxy-7-methoxy-flavonone, naringenin, apiflorol and / or its combinations. Active fractions according to Claim 4, characterized in that the active fractions of ethyl acetate and / or chloroform comprise 0.2% MIC. Active fractions according to claims 1 to 5, characterized in that they comprise cosmetic and / or pharmaceutical formulations. PHARMACEUTICAL AND / OUCOSMETIC COMPOSITIONS according to Claim 6, characterized in that the formulations comprise dosage forms such as emulsions, gels, creams, ointments, solutions and / or shampoos. PHARMACEUTICAL AND / OUCOSMETIC COMPOSITIONS according to Claim 7, characterized in that the formulations preferably comprise creams, exampus gels. PHARMACEUTICAL AND / OUCOSMETIC COMPOSITIONS according to Claim 8, characterized in that all of the cream formulations comprise the components chosen as self-emulsifying and / or surfactant, e.g. optionally, polymeric consistency agents such as glyceryl omostearate, 2% w / v hydroxyethyl cellulose dispersion, such as emollient, mineral oil, cetyl acetate and / or lanolinacetyl alcohol, volatile silicone, 2-octyldodecanol; as a humectant, propylene glycol and / or glycerin; and preservatives such as parabens. PHARMACEUTICAL AND / OUCOSMETIC COMPOSITIONS according to claim 8, characterized in that all of the gel formulations comprise selected components of polymeric consistency and / or gelling agents, for example carbonyl polymers, hydroxychellulose; acidifying and / or alkaline agents such as 20% (w / v) sodium hydroxide solution, triethanolamine; surfactants such as cetostearyl alcohol, ethoxylated and / or propoxylated ethyl alcohol, fatty acid ester polyol as a wetting agent such as propylene glycol, sorbitol 70% and / or glycerin; preservatives such as parabens. PHARMACEUTICAL AND / OUCOSMETIC COMPOSITIONS according to Claim 8, characterized in that the shampoo formulations comprise selected anionic, cationic and / or amphoteric primary, and / or secondary amphoteric components such as sodium lauryl ether sulfate, lauryl sulfate detriethanolamine, disodium lauryl sulfosuccinate and / or disodium lauryl sulfate, coconut fatty acid diethanolamide, propyl betaine cocoamido, 3-oleyl phosphate; neutralizing agent, such as 10% (w / v) citric acid solution; consistency agent such as glycol distearate, 20% (w / v) sodium chloride solution; and preservatives such as osparabens. Use of extracts according to Claims 1 to 11, characterized in that it is for the preparation of pharmaceutical and / or cosmetic decompositions as antimicrobial. Use of extracts according to Claim 12, characterized in that the application as an antimicrobial comprises the use as a preservative in cosmetic and / or pharmaceutical formulations. 14. USE OF EXTRACTS according to claims 12 and 13, characterized in that it has an antimicrobial activity comprising the action on gram-negative, gram-positive, yeast and / or fungal bacteria (mold).