PT106708A - APPLICATION OF SUMMARY EXTRACTS AS ANTIMICROBIAL AGENT - Google Patents

Abstract

The present invention relates to biosurfactant or BIOSTATIC COMPOSITIONS OF RHUS CORIARIA L. LEAVES, (SUMMER) EXTRACTED THROUGH ETHANOL. EXTRACT PRESENTED ANTIMICROBIAL ACTIVITY AGAINST VARIOUS MICRO-ORGANISMS ASSOCIATED WITH ANIMAL PLANTS AND FOOD CONTAMINATION, SUCH AS BACILIUS CEREUS, BACILIUS MYCOIDES, BACILIUS SUBTILIS, CANDIDA SUBTILIS, CANDIDA ALBICANS ATCC 90028, CANDIDA PARAPSILOSIS (ATCC 22019), ENTEROCOCCUS SP., ESCHERICIA COLI (ISOLATED AT UTAD), ESCHERICHIA COLI ATCC 25922, AMONG OTHERS. IN A GENERAL MODE, THIS EXTRACT REVEALED TO BE OF GREAT UTILITY IN THE AGROFINDER INDUSTRY AND THE BIOLOGICAL PROTECTION OF CULTURES.

Description

DESCRIPTION " Application of sumac extracts as an antimicrobial agent "

Technical Domain of the Invention

This invention relates to biocidal or biostatic compositions extracted from Rhus coriaria L. leaves which have been active against various microorganisms such as Bacillus cereus, Bacillus mycoides, Bacillus subtilis, Candida subtilis, Candida albicans ATCC 90028, Candida parapsilosis (ATCC 22019), Enterococcus sp Eschericia coli (isolated from UTAD), Escherichia coli ATCC 25922, Fusarium sp. among other pathogens. These compositions have proven to be very useful in combating fungal and bacterial infections that cause disease in plants and food. FIELD OF THE INVENTION The present invention is in the area of biocidal or biostatic compositions resulting from the ethanolic extraction of leaves of R. coriaria L.

Background of the Invention The base of this invention is the preparation of a natural extract obtained through the leaf of a plant commonly known as sumac, abundant in the Douro region. Some documents analyzed showed that the use of extracts derived from plants allowed to combat the infection promoted by some fungi and pathogenic bacteria in cultures (eg: vines, potatoes, prunodes, pomegranates, horticultural, ornamental and lawn) 1/8 and to combat contaminations in foods at the level of the food industry.

To obtain the sumac extract, R. coriaría leaves from the Douro region were collected and dried. Then the material was ground and a mixture of 20 g of ground material was prepared in 80 ml of 100% ethanol. The mixture was stirred for 48 h, filtered and evaporated at 80 ° C on a rotary evaporator. The resulting viscous liquid was resuspended in 50 ml of lyophilized distilled water. The resulting lyophilization product was subsequently resuspended in DMSO (10 mg / ml) and filtered through a sterile 0.22 μm filter. The extract was collected in a sterile vial and stored in the refrigerator for further use. In order to evaluate the antimicrobial activity of the extract, 15 μΐ of extract were impregnated in sterile and blank discs. After a drying period of 5 minutes, these were placed in Petri dishes previously cultured with different microorganisms.

Comparing the method used in this work with the documents indicated by the state of the art, there are some differences. In Adwan et al. (2010), the extract and its preparation were different from that described in this invention, the only similarity being the type of solvent used for the extraction. For the preparation of the extract Adwan et al. used 30/50 g of dry sumac seeds in 200/300 ml of 80% ethanol. The mixture was filtered through filter paper and allowed to dry at 37 ° C. At the time of use, the extract was resuspended in distilled water at a concentration of 100 mg / ml.

With regard to the work carried out by Sá (2010), the plant used was different from the one used in this invention, as well as the method of extracting and preparing the extract. The extract made by Sá was prepared by a 2/8 mixture of 2g of Arbutus unedo leaves in 250 ml of hot water for 45 minutes. The extract was filtered through filter paper, frozen and lyophilized. At its use the extract was resuspended in water at a concentration of 20 mg / ml.

When analyzing other available documents there are still some differences, such as the type of solvent used, the method and the type of material being extracted. These points can be verified for example in the work of Nasar-Abbas (2004), in which they used sumac fruits, placed in a mixture at the rate of 5g of fruits in 95 ml of distilled water. This mixture was allowed to stir for 1 hour at room temperature, boiled for 2 minutes and filtered through filter paper.

The differences demonstrated by comparing our method with the work of Adwan et al. (2010), Sá (2010) and Nasar-Abbas (2004), as well as other studies, reinforce the innovative character of this product. 3/8

SUMMARY OF THE INVENTION

In this invention, ethanolic extracts of sumac leaves (Rhus coriaria L.) were prepared in order to evaluate their activity against various pathogenic microorganisms of animal and food plants. The extracts were tested on Bacillus cereus, Bacillus mycoides, Bacillus subtilis, Candida albicans (ATCC 90028), Candida parapsilosis (ATCC 22019), Enterococcus sp., Escherichia coli (UTAD isolated), Escherichia coli (ATCC 25922), Fusarium sp. and other pathogenic microorganisms. It was found that R. coriaria extracts showed efficacy against all microorganisms tested, with the microorganism being more susceptible C. parapsilosis (HIM = 18.75 mm) while the less susceptible was B. subtilis (HIM = 11.35 mm ). By observing the different values of inhibition obtained, the microorganisms Enterococcus sp. and C. parapsilosis as the most susceptible to the extract and the B. cereus, B. subtilis, B. mycoides and E. coli groups of UTAD as the least susceptible.

Description of the drawings:

Figure 1: Inhibition halos resulting from the application of sumac leaf extract in Candida parapsilosis (left) and Enterococcus sp. (right).

Figure 2: Inhibition hairs resulting from the application of sumac leaf extract in Bacillus subtilis (left) and Bacillus mycoides (right).

Figure 3: Mean inhibition halos ordered by increasing order of inhibition values (mean and standard error). 4/8

Figure 4: Chromatogram of flavonoid analysis (355 nm) detected in the analysis of sumac extract by HPLC.

Figure 5: Chromatogram of the analysis of the derivatives of quercitina (280 nm) detected in the analysis of the sumac extract by HPLC.

Detailed description of the invention

Plants are an important source of compounds with biological activity with great scientific and economic interest that contribute to food, health and improvement of the quality of life of humanity. For this reason its use and its secondary compounds are very frequent in very varied industries such as furniture, food, cosmetics, pharmaceutical industry, among others.

A Mediterranean shrub that proved potentially interesting was Rhus coriaria L., commonly known as sumac. It is a shrub or small deciduous tree that can reach 3-4 meters high belonging to the family of Anacardiaceae, botanical family of plants rich in resins and tannins. The sumac is widely distributed in the Canary Islands and throughout the Mediterranean zone covering Iran and Afghanistan. In Portugal it can easily be found from Trás-os-Montes (Terra Quente and Douro valley) to the Algarve and also on the islands (Madeira and Azores), on slopes and on the edges of woods, paths or fields of other cultures, such as vineyards and orchards This species was once widely used in tanning and dyeing industries, due to the high presence of secondary compounds, namely tannins (20-30%), in the leaves and bark of the stem. It should be noted that the amount of tannins may vary depending on the vegetative period 5/8 of the plant, the species and the variety. In this work an ethanolic extract of leaves of this species was prepared, in order to evaluate its action against several pathogenic microorganisms of crops and foods. The extract was tested on two Gram-negative bacteria (Escherichia coli (UTAD isolate) and Escherichia coli (ATCC 25922), four Gram positive bacteria (Bacillus cereus, Bacillus mycoides, Bacillus subtilis and Enterococcus sp.) And two yeasts (Candida albicans ATCC 90028), Candida parapsilosis (ATCC 22019).

Obtaining the extract:

The leaves of R. coriaria were previously harvested, in the region of Vila Real, cleaned and placed to dry in an oven, with air circulation, at 45 ° C, for one week. The dried material was then milled in a mill. 20 g of the milled material and 80 ml of 100% ethanol were placed in an Erlenmayer flask. The mixture was stirred for 48 h, filtered through filter paper and evaporated on a rotary evaporator at 80 ° C to remove all the ethanol present. The resulting viscous liquid was diluted in 50 ml of distilled water, frozen and lyophilized for one week. The resulting lyophilization product was resuspended in DMSO (10 mg / ml) and filtered through a sterile filter of 0.22 μm porosity. The extract was collected in a sterile vial and stored in the refrigerator for use.

Antimicrobian activity:

Antimicrobial activity evaluation: With the aid of a loop, colonies of microorganisms were removed from plaques previously cultured and suspended in 9 ml of sterile water. The suspensions were compared with the 6/8 scale of 0.5 and 1 of McFarland for yeasts and bacteria, respectively, according to NCCLS standards. Then, the microorganisms were seeded through sterile swabs in plates with the Mueller Hinton culture medium (MH), previously identified. For each microorganism 3 replicates were performed.

Preparation of discs impregnated with extract: The sterilized and blank discs were impregnated with 15 μl of extract, allowed to dry 5 minutes and placed in the previously seeded Petri dishes. Plates were incubated in a forced air oven at 36 ° C for 48 h. At the end of the incubation period, the inhibition halos were measured with a digital meter and the results recorded on a table.

Statistical analysis: The factorial analysis of variance (ANOVA-factorial) was used to evaluate the antimicrobial activity in the STATISTICA version 9.0 program, and the null hypothesis was rejected when P < 0.05.

Results:

Table 1 shows the results obtained from the application of sumac leaf extract against the microorganisms under study.

Microorganism Mean (mm) DP B. cereus 11.98 0.43 B. subtilis 11.35 1.57 B. mycoides 11.44 0.91 E. coli UTAD 11.47 0.81 E. coli ATCC 25922 15, 16 0.77 Enterococcus sp. 18.73 1.40 C.parapsilosis ATCC 22019 18.75 2.78 C. albicans ATCC 90028 15.34 1.31

Table 1. Susceptibility of different microorganisms to sumac extracts (mean and standard deviation (SD) of n = 9). 7/8

In general, it was possible to verify that the microorganisms most susceptible to the extract were Candida parapsilosis and Enterococcus sp. (Figure 1) and the less susceptible were B. subtilis and B. mycoides (Figure 2).

By the analysis of Figure 3 we can verify the existence of three distinct groups regarding the differences of susceptibility existing in the microorganisms for this vegetal extract. A first group formed by the microorganisms that presented the largest inhibition halos (Enterococcus sp. And C. parapsilosis), the second group with intermediate susceptibility (E. coli ATCC 25922 and C. albicans) and the last group consisting of the microorganisms where the lowest inhibition values were obtained (B. cereus, B. subtilis, B. mycoides and E. coli UTAD).

Crude extract analysis: The extract was analyzed by high performance liquid chromatography (HPLC) from which it was possible to verify that the sumac compound has a large amount of flavonoids (Figure 4), mainly quercetin derivatives (Figure 5). This plant further shows other phenolic compounds such as tannins.

Vila Real, December 18, 2013 8/8

Claims (11)

A sumac extract for application as an antimicrobial agent characterized in that it is obtained from sumac leaves by reaction carried out by a polar solvent. Sumac extract for application as an antimicrobial agent according to claim 1, characterized in that the extract has phenolic compounds with antibacterial and antifungal action. A sumac extract for application as an antimicrobial agent according to claim 2, characterized in that the phenolic compounds belong to the class of flavonoids. A sumac extract for application as an antimicrobial agent according to the preceding claims characterized in that the flavonoids are tannins and quercetin derivatives. A sumac extract as an antimicrobial agent according to the preceding claims characterized in that its composition is biocidal or biostatic of Gram negative and Gram positive bacteria. A sumac extract as an antimicrobial agent according to the preceding claims characterized in that quercetin has the ability to cross the bacterial wall and to lyse the membrane causing increased toxicity within the cell. A sumac extract for application as an antimicrobial agent according to claim 5, characterized in that Gram negative are Escherichia coli (ATCC 25922) and Escherichia coli (isolated in UTAD). 1/2 A sumac extract for application as an antimicrobial agent according to claim 5, characterized in that the Gram positive are Enterococcus spp., Bacillus cereus, Bacillus mycoides and Bacillus subtilis. A sumac extract for application as an antimicrobial agent according to claim 2, characterized in that it is biocidal or biostatic of yeasts and molds. A sumac extract as an antimicrobial agent according to the preceding claims characterized in that quercetin has the ability to cross the bacterial wall and to lyse the membrane causing increased toxicity within the cell. A sumac extract for application as an antimicrobial agent according to claim 9, characterized in that the yeasts and molds are Candida parapsilosis (ATCC 22019), Candida albicans (ATCC 90028) and Fusarium sp. Vila Real, December 18, 2013 2/2