FARMACIA, 2015, Vol. 63, 2 ORIGINAL ARTICLE CONTRIBUTIONS TO THE ANTIMICROBIAL AND ANTIFUNGAL STUDY OF THE AQUEOUS EXTRACT OF PRUNUS SPINOSA L.

GABRIELA GEGIU¹*, ANDREI-DAN BRANZA1, LAURA BUCUR2, MIRCEA GRIGORIAN3, TRAIAN TACHE4, VICTORIA BADEA1

¹Department of Microbiology, Ovidius University Constanta, Faculty of Dental Medicine, 7 Ilarie Voronca Street, Constanta, Romania ²Department of Pharmacognosy, Ovidius University Constanta, Faculty of Pharmacy, 1 University Street, Campus, Building B, Constanta, Romania 3Department of Physiology, Ovidius University Constanta, Faculty of Dental Medicine, 7 Ilarie Voronca Street, Constanta, Romania 4Department of Semiology, Ovidius University Constanta, Faculty of Pharmacy, 1 University Street, Campus, Building B, Constanta, Romania Department of Microbiology, Ovidius University Constanta, Faculty of Dental Medicine, 7 Ilarie Voronca Street, Constanta, Romania

*corresponding author: [email protected] Manuscript received: October 2013

Abstract The aim of this study was to evaluate the antibacterial and antifungal action of aqueous extracts from Prunus spinosa L. dried fruit determined on five bacterial strains and one fungal strain. The products taken into discussion have been harvested from two geographical regions. There have been prepared two aqueous extracts in different dilutions. The antibacterial and antifungal activity has been assessed using the disc diffusion method. The results obtained demonstrate that the solutions tested do not have antifungal activity, but at the same time, our study proves that the Staphylococcus aureus and Escherichia coli strains are sensitive to these solutions. In conclusion we can state that the two aqueous extracts from the Prunus spinosa L. species, obtained from different geographical areas, may be used for the future development of new pharmaceutical products.

Rezumat Lucrarea îşi propune evaluarea acţiunii antibacteriene şi antifungice a extractelor apoase ale fructelor uscate de Prunus spinosa L. determinată pe cinci tulpini bacteriene şi una fungică. Produsul vegetal luat în lucru a fost recoltat din două regiuni geografice. Au fost preparate două extracte apoase în diluţii diferite. Activitatea antibacteriană şi antifungică a fost evaluată utilizând metoda difuzimetrică. Rezultatele obţinute demonstrează că soluţiile testate nu au activitate antifungică; în acelaşi timp, studiul nostru dovedește că tulpinile de Staphylococcus aureus şi Escherichia coli sunt sensibile la aceste soluții. În concluzie putem afirma că cele două extracte apoase de la specia Prunus spinosa L., provenite din zone geografice diferite, pot fi utilizate pentru a dezvolta în viitor produse farmaceutice noi.

Keywords: Prunus spinosa L., aqueous extracts, antibacterial activity

Introduction still empirical, even if their potential is known from ancient times [3]. Therapeutically practice using active substances The pharmaceutical form that can be used under biosynthesized with the help of plants defines various ways (infusions, decoctions, baths, phytotherapy, an old branch of contemporary cataplasm, syrups, inhalations, ointments, capsules, therapeutics, with a range and tradition from ancient tinctures) must be standardized and adequate to times [2]. The vegetal products are indicated in their posology [2]. chronic diseases as well as in acute disorders [2]. Currently, clinicians around the world are facing a There must be respected and well known the serious problem of increasing bacterial resistance to following: the method of administration, the antibiotics [6]. duration of the treatment and also when the plants Traditionally, the susceptibility of bacteria to anti- are due to be collected, so that the therapeutic microbial agents is assessed by measuring the effects will be the expected ones. In addition, the Inhibitory Concentration 50 (IC50) and the usage of the natural compounds from the plants is Minimal Inhibitory Concentration (MIC) [6].

275 FARMACIA, 2015, Vol. 63, 2 One of the most important directions of research drying is complete once the fruit gets wrinkled. related to bacterial resistance is represented by the After the harvesting and drying, the product was sorted. discovery of new compounds with new structures To carryout microbiological studies from the dry and new mechanisms of action, which will prevent vegetal product Prunus spinosae fructus, there have the occurrence and development of bacteria been prepared two aqueous extracts in dilutions of resistance to antibiotics [17]. 1:10 and 5:10, from each geographical area: Argeş Prunus spinosa L. (blackthorn) belongs to the and Tulcea. Subregnum Cormobionta, Phylum Magnoliophyta, Obtaining solutions 10% and 50% Class Magnoliatae, Order Rosales, Family Rosaceae, We detached the pulp from the kernel and weighed Subfamily Prunoidae [7, 15]. It is a thorny, 1 gram for the 10% solution or 5 grams for the 50% indigenous; 1-3 m tall shrub that can be found from solution. The vegetal product was then distributed the plains to the mountain level, in all regions of the in two test tubes of 20 mL. After that, 10 mL of country such as rocky hills, rocks and sunny coasts water were poured over the product. The tubes were and usually it can be found at forest edges [12]. kept in boiling water bath for 30 minutes; the Prunus spinosa L. has branches that end in thorns mixture was filtered through a filter paper and made of 4-8 centimetres. Its stem is covered by a rifted, up to 10 mL by washing the residue with water [4]. grey bark. The flowers are solitary, long petiolated, Microbiological studies with white petals and numerous stamina, with red The research were conducted on bacterial and or yellow antenna (which appear before the leaves), fungal strains reference ATCC (American Type of and they have a characteristic smell. Sometimes, Culture Collection) lyophilized, stabilized and the leaves are hairy on the inner side, denticulate viable (each pellet used contained the lyophilized and oval. The fruits are juicy and fleshy, with micro-organism, which was distributed in an central kernels; spherical and green until maturity, environment well studied, to ensure the viability when the colour is becoming dark-blue. Regarding and stability of results in time), obtained from Culti the taste, this is sour, sweet and very astringent [14]. Control from Liofilchem, Italy. In the composition of flowers there are flavonoids Under aseptic conditions each pellet was immersed (including quercetin, kaempferol), organic acids, in a container with 1-2 mL of the liquid culture magnesium and potassium salts [12]. In empirical (triptych soy bullion for aerobically bacteria and medicine, its properties are weakly laxative, tonic, Candida yeasts). After that, the container was diuretics and more recently antihypertensive [1]. incubated at 36º±1ºC, for 5-10 minutes. From the The essential components of the fruit are sugars, content of the recipient a drop of the suspension salts of calcium and Magnesium, vitamin C, was inoculated on the surface which contained a tannins, organic acids, prunicianine, anthocyanin, culture environment suitable for the growth of each polyphenols and gumiresines [12]. The fruits are type of micro-organism used in the study. It was used in biliary dyskinesia and renal diseases, but subsequently incubated under appropriate they also have very good astringent results if they temperature and atmospheric conditions, for 24-96 are used in the form of decoct in diarrheic states [1]. hours. We used the following reference strains: Staphylococcus ATCC 25923, Streptococcus Materials and Methods ATCC 19615, Enterococcus ATCC 19433, Escherichia coli ATCC 25922, Pseudomonas Harvesting the plant and obtaining the aqueous ATCC 27853, Candida albicans ATCC 10231. extract The principle of the method and sensitivity test for the We used dried fruit pulp from Prunus spinosa L. concerned substances, on bacterial and fungal strains species, harvested from Argeş and Tulcea areas, The antimicrobial activity was determined by the from Romania. The harvesting has been done in the disc diffusion method [5]. The principle of this first two weeks of September, because in fall the method consists in the contact of a standard content of tannins is higher. It is highly medium from the microorganism-test with recommended that the harvesting shall take place increasing concentrations of the test sample. after the rime has fallen, because the astringency is Microorganism-suspension test: in the isotonic lost and the sugar percentage is increased. The sodium chloride solution, suspensions have been fruits were collected manually, fruit by fruit. They prepared with a concentration of 107 colony were picked up in a fresh condition and were dried forming units/mL micro-test, corresponding to the at a temperature of approximately 35°C, using a tube’s turbidity of 0.5 Mac Farland [5]. desiccator (it is indicated an optimum temperature Microorganism-suspension tests were inoculated on because at the sudden rise of the temperature, the the following culture media: Blood-Agar, Muller- fruits will lose their natural colour, the sugars shall Hinton, Sabouraud Dextrose Agar. On the surface be deposed at the surface, the colour becoming of the media, discs of sterile filter paper were whitish and also caramelisation can appear). The placed, soaked with 10 mL of the test products. The 276 FARMACIA, 2015, Vol. 63, 2 Petri dishes have then been incubated at 37°C, 24 Results and Discussion hours for bacteria and at 35°C, 24 hours for fungus The diameter of the inhibition zone was measured [5]. After that we examined the Petri dishes, by for each dilution and was expressed in millimetres. recording the diameter of the inhibition zones for The obtained results are shown in Table I and Table II. each microorganism and for each dilution of the test sample. Table I The diameters of the inhibition zones on bacterial and fungal cultures for the Argeş sample. STUDIED MICROORGANISMS

coli coli 10231 Candida Candida SAMPLE Escherichia Escherichia ATCC25922 ATCC 19615 ATCC 19433 ATCC 27853 ATCC Enterococcus Enterococcus Streptococcus Streptococcus Pseudomonas Pseudomonas Staphylococcu s ATCCs 25923 albicans ATCC albicans Diameters of the inhibition zones (mm) Argeș 10% 10 mm 12 mm resistance 12 mm resistance resistance Argeș 50% 12 mm 13 mm resistance 12 mm 8 mm resistance Table II The diameters of the inhibition zones on bacterial and fungal cultures for the Tulcea sample. STUDIED MICROORGANISMS

coli coli 10231 Candida Candida SAMPLE Escherichia Escherichia ATCC25922 ATCC 25923 ATCC 19615 ATCC 19433 ATCC 27853 ATCC Enterococcus Enterococcus Streptococcus Streptococcus Pseudomonas Pseudomonas albicans ATCC albicans Staphylococcus Staphylococcus Diameters of the inhibition zones (mm) Tulcea 10% 11 mm 12 mm resistance 13 mm resistance resistance Tulcea 50% 14 mm 14 mm 12 mm 12 mm resistance resistance Resistance mutation occur

By using the disc diffusion method our results The antimicrobial activity of the studied aqueous demonstrated that the tested solutions have extracts is differentiated according to the different antibacterial activity, but they don’t have concentration of the substance, but also to the area antifungal activity. This fact was demonstrated by of origin, such as: the extract from Tulcea area 10% total resistance of Candida albicans strains for both has an antibacterial effect against Staphylococcus concentrations of the solutions obtained from plants sp., the inhibitory effect being of similar intensity harvested from the two areas (Figure 1). to the one shown in Argeş county area for the same concentration. The extract from Tulcea area 50% had a slightly higher sensitivity (14 mm) compared with that from Argeş 50% (12 mm) as shown in Figure 2.

Figure 1. Figure 2. The action of the aqueous extracts (10% and 50%) The action of the aqueous extracts (10% and 50%) of Prunus spinosa L. on Candida albicans sp. of Prunus spinosa L. on Staphylococcus sp.

277 FARMACIA, 2015, Vol. 63, 2 The results obtained by testing the sensitivity effect presented moderate antibacterial activity (8 mm) as of Streptococcus sp. strain to the solutions presented in Figure 5. demonstrated that with the increase of solution concentration, it increases the diameter of the inhibition zones on bacterial culture (Figure 3).

Figure 5. The action of the aqueous extracts (10%, 50%) of Prunus spinosa L. from Argeș County on Pseudomonas sp.

A. The results of our study show that Escherichia coli sp. is sensitive to both concentrations of the solutions obtained from plants harvested from the two areas (Figure 6).

B. Figure 3. The actions of Prunus spinosa L. aqueous extracts (10% and 50%) from Tulcea (A) compared with the ones from Argeş (B) on Streptococcus sp.

Enterococcus sp. was resistant to both concentrations obtained from both areas, except Tulcea 50% extract, where initially was an area of Figure 6. growth inhibition for the bacteria colonies, The action of the aqueous extracts (10% and 50%) followed by the appearance of resistance mutations of Prunus spinosa L. on Escherichia coli sp. in the inhibition area, as seen in Figure 4. There are data published in literature about similar studies performed on other types of aqueous extracts, such as those obtained from the stems of Ribes nigrum and the layer of Rosmarinus officinalis, which demonstrate the presence of antibacterial and antifungal activity like the ones achieved in our study; there is similarity regarding the presence of active principles in the aqueous extract of Prunus spinosa L. and the ones obtained from the plants used in the above mentioned studies, respectively: flavonoids, quercetin, myricetin, kaempferol, polyphenolic acids [3, 8, 9, 13]. The disc diffusion method applied in our study has been used also by other authors [10, 11, 13, 14] for the determination of antibacterial and antifungal Figure 4. action of extracts obtained from plants or from The action of the aqueous extracts (10%, 50%) of volatile oils. In the group of Gram positive bacteria, Prunus spinosa L. on Enterococcus sp. the highest level of resistance has been recorded for

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