FARMACIA, 2015, Vol.

63, 2

ORIGINAL ARTICLE

CONTRIBUTIONS TO THE ANTIMICROBIAL AND ANTIFUNGAL

STUDY OF THE AQUEOUS EXTRACT OF PRUNUS SPINOSA L.
GABRIELA GEGIU*, ANDREI-DAN BRANZA1, LAURA BUCUR2, MIRCEA GRIGORIAN3,
TRAIAN TACHE4, VICTORIA BADEA1
Department of Microbiology, Ovidius University Constanta, Faculty of Dental Medicine, 7 Ilarie Voronca Street, Constanta,
Romania
Department of Pharmacognosy, Ovidius University Constanta, Faculty
of Pharmacy, 1 University Street, Campus, Building B, Constanta, Romania
3
Department of Physiology, Ovidius University Constanta, Faculty of Dental Medicine, 7 Ilarie Voronca Street, Constanta,
Romania
4
Department 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: gegiugabriela@yahoo.com
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 i propune evaluarea aciunii antibacteriene i antifungice a extractelor apoase ale fructelor uscate de Prunus
spinosa L. determinat pe cinci tulpini bacteriene i una fungic. Produsul vegetal luat n lucru a fost recoltat din dou regiuni
geografice. Au fost preparate dou extracte apoase n diluii diferite. Activitatea antibacterian i antifungic a fost evaluat
utiliznd metoda difuzimetric.
Rezultatele obinute demonstreaz c soluiile testate nu au activitate antifungic; n acelai timp, studiul nostru dovedete c
tulpinile de Staphylococcus aureus i Escherichia coli sunt sensibile la aceste soluii. n concluzie putem afirma c cele dou
extracte apoase de la specia Prunus spinosa L., provenite din zone geografice diferite, pot fi utilizate pentru a dezvolta n
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].
The pharmaceutical form that can be used under
various ways (infusions, decoctions, baths,
cataplasm, syrups, inhalations, ointments, capsules,
tinctures) must be standardized and adequate to
their posology [2].
Currently, clinicians around the world are facing a
serious problem of increasing bacterial resistance to
antibiotics [6].
Traditionally, the susceptibility of bacteria to antimicrobial agents is assessed by measuring the
Inhibitory Concentration 50 (IC50) and the
Minimal Inhibitory Concentration (MIC) [6].

Therapeutically practice using active substances

biosynthesized with the help of plants defines
phytotherapy, an old branch of contemporary
therapeutics, with a range and tradition from ancient
times [2]. The vegetal products are indicated in
chronic diseases as well as in acute disorders [2].
There must be respected and well known the
following: the method of administration, the
duration of the treatment and also when the plants
are due to be collected, so that the therapeutic
effects will be the expected ones. In addition, the
usage of the natural compounds from the plants is
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One of the most important directions of research

related to bacterial resistance is represented by the
discovery of new compounds with new structures
and new mechanisms of action, which will prevent
the occurrence and development of bacteria
resistance to antibiotics [17].
Prunus spinosa L. (blackthorn) belongs to the
Subregnum Cormobionta, Phylum Magnoliophyta,
Class Magnoliatae, Order Rosales, Family Rosaceae,
Subfamily Prunoidae [7, 15]. It is a thorny,
indigenous; 1-3 m tall shrub that can be found from
the plains to the mountain level, in all regions of the
country such as rocky hills, rocks and sunny coasts
and usually it can be found at forest edges [12].
Prunus spinosa L. has branches that end in thorns
of 4-8 centimetres. Its stem is covered by a rifted,
grey bark. The flowers are solitary, long petiolated,
with white petals and numerous stamina, with red
or yellow antenna (which appear before the leaves),
and they have a characteristic smell. Sometimes,
the leaves are hairy on the inner side, denticulate
and oval. The fruits are juicy and fleshy, with
central kernels; spherical and green until maturity,
when the colour is becoming dark-blue. Regarding
the taste, this is sour, sweet and very astringent [14].
In the composition of flowers there are flavonoids
(including quercetin, kaempferol), organic acids,
magnesium and potassium salts [12]. In empirical
medicine, its properties are weakly laxative, tonic,
diuretics and more recently antihypertensive [1].
The essential components of the fruit are sugars,
salts of calcium and Magnesium, vitamin C,
tannins, organic acids, prunicianine, anthocyanin,
polyphenols and gumiresines [12]. The fruits are
used in biliary dyskinesia and renal diseases, but
they also have very good astringent results if they
are used in the form of decoct in diarrheic states [1].

drying is complete once the fruit gets wrinkled.

After the harvesting and drying, the product was sorted.
To carryout microbiological studies from the dry
vegetal product Prunus spinosae fructus, there have
been prepared two aqueous extracts in dilutions of
1:10 and 5:10, from each geographical area: Arge
and Tulcea.
Obtaining solutions 10% and 50%
We detached the pulp from the kernel and weighed
1 gram for the 10% solution or 5 grams for the 50%
solution. The vegetal product was then distributed
in two test tubes of 20 mL. After that, 10 mL of
water were poured over the product. The tubes were
kept in boiling water bath for 30 minutes; the
mixture was filtered through a filter paper and made
up to 10 mL by washing the residue with water [4].
Microbiological studies
The research were conducted on bacterial and
fungal strains reference ATCC (American Type of
Culture Collection) lyophilized, stabilized and
viable (each pellet used contained the lyophilized
micro-organism, which was distributed in an
environment well studied, to ensure the viability
and stability of results in time), obtained from Culti
Control from Liofilchem, Italy.
Under aseptic conditions each pellet was immersed
in a container with 1-2 mL of the liquid culture
(triptych soy bullion for aerobically bacteria and
Candida yeasts). After that, the container was
incubated at 361C, for 5-10 minutes. From the
content of the recipient a drop of the suspension
was inoculated on the surface which contained a
culture environment suitable for the growth of each
type of micro-organism used in the study. It was
subsequently
incubated
under
appropriate
temperature and atmospheric conditions, for 24-96
hours. We used the following reference strains:
Staphylococcus ATCC 25923, Streptococcus
ATCC 19615, Enterococcus ATCC 19433,
Escherichia coli ATCC 25922, Pseudomonas
ATCC 27853, Candida albicans ATCC 10231.
The principle of the method and sensitivity test for the
concerned substances, on bacterial and fungal strains
The antimicrobial activity was determined by the
disc diffusion method [5]. The principle of this
method consists in the contact of a standard
medium from the microorganism-test with
increasing concentrations of the test sample.
Microorganism-suspension test: in the isotonic
sodium chloride solution, suspensions have been
prepared with a concentration of 107 colony
forming units/mL micro-test, corresponding to the
tubes turbidity of 0.5 Mac Farland [5].
Microorganism-suspension tests were inoculated on
the following culture media: Blood-Agar, MullerHinton, Sabouraud Dextrose Agar. On the surface
of the media, discs of sterile filter paper were
placed, soaked with 10 mL of the test products. The

Materials and Methods

Harvesting the plant and obtaining the aqueous
extract
We used dried fruit pulp from Prunus spinosa L.
species, harvested from Arge and Tulcea areas,
from Romania. The harvesting has been done in the
first two weeks of September, because in fall the
content of tannins is higher. It is highly
recommended that the harvesting shall take place
after the rime has fallen, because the astringency is
lost and the sugar percentage is increased. The
fruits were collected manually, fruit by fruit. They
were picked up in a fresh condition and were dried
at a temperature of approximately 35C, using a
desiccator (it is indicated an optimum temperature
because at the sudden rise of the temperature, the
fruits will lose their natural colour, the sugars shall
be deposed at the surface, the colour becoming
whitish and also caramelisation can appear). The
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Petri dishes have then been incubated at 37C, 24

hours for bacteria and at 35C, 24 hours for fungus
[5]. After that we examined the Petri dishes, by
recording the diameter of the inhibition zones for
each microorganism and for each dilution of the
test sample.

Results and Discussion

The diameter of the inhibition zone was measured
for each dilution and was expressed in millimetres.
The obtained results are shown in Table I and Table II.

Table I
The diameters of the inhibition zones on bacterial and fungal cultures for the Arge sample.

Arge 10%
Arge 50%

10 mm
12 mm

Candida
albicans ATCC
10231

Pseudomonas
ATCC 27853

Escherichia
coli
ATCC25922

Enterococcus
ATCC 19433

Streptococcus
ATCC 19615

SAMPLE

Staphylococcu
s ATCC 25923

STUDIED MICROORGANISMS

Diameters of the inhibition zones (mm)

12 mm
resistance
12 mm
resistance
13 mm
resistance
12 mm
8 mm

resistance
resistance

Table II
The diameters of the inhibition zones on bacterial and fungal cultures for the Tulcea sample.

Tulcea 10%
Tulcea 50%

11 mm
14 mm

12 mm
14 mm

Diameters of the inhibition zones (mm)

resistance
13 mm
resistance
12 mm
12 mm
resistance
Resistance mutation occur

Candida
albicans ATCC
10231

Pseudomonas
ATCC 27853

Escherichia
coli
ATCC25922

Enterococcus
ATCC 19433

Streptococcus
ATCC 19615

Staphylococcus
ATCC 25923

SAMPLE

STUDIED MICROORGANISMS

resistance
resistance

By using the disc diffusion method our results

demonstrated that the tested solutions have
different antibacterial activity, but they dont have
antifungal activity. This fact was demonstrated by
total resistance of Candida albicans strains for both
concentrations of the solutions obtained from plants
harvested from the two areas (Figure 1).

The antimicrobial activity of the studied aqueous

extracts is differentiated according to the
concentration of the substance, but also to the area
of origin, such as: the extract from Tulcea area 10%
has an antibacterial effect against Staphylococcus
sp., the inhibitory effect being of similar intensity
to the one shown in Arge county area for the same
concentration. The extract from Tulcea area 50%
had a slightly higher sensitivity (14 mm) compared with
that from Arge 50% (12 mm) as shown in Figure 2.

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

Figure 2.
The action of the aqueous extracts (10% and 50%)
of Prunus spinosa L. on Staphylococcus sp.
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The results obtained by testing the sensitivity effect

of Streptococcus sp. strain to the solutions
demonstrated that with the increase of solution
concentration, it increases the diameter of the
inhibition zones on bacterial culture (Figure 3).

presented moderate antibacterial activity (8 mm) as

presented in Figure 5.

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

A.

B.

Figure 3.
The actions of Prunus spinosa L. aqueous extracts
(10% and 50%) from Tulcea (A) compared with the
ones from Arge (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
growth inhibition for the bacteria colonies,
followed by the appearance of resistance mutations
in the inhibition area, as seen in Figure 4.

Figure 6.
The action of the aqueous extracts (10% and 50%)
of Prunus spinosa L. on Escherichia coli sp.
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
action of extracts obtained from plants or from
volatile oils. In the group of Gram positive bacteria,
the highest level of resistance has been recorded for
the Enterococcus species and from the group of
Gram negative bacteria for the Pseudomonas
species, both species with established and
demonstrated resistance to many antibiotics.
Differences in the antibacterial action of the
solutions with identical concentration can be

Figure 4.
The action of the aqueous extracts (10%, 50%) of
Prunus spinosa L. on Enterococcus sp.
For Pseudomonas sp. it was noted total resistance
for 10% concentrations obtained from both areas
and for the 50% extract from Tulcea area, while the
50% concentration extract from Arge County
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explained by the fact that the properties of the soil

in which the same plant develops, may be different
because of the proportion of certain elements from
its composition. For example, regarding
Pseudomonas sp., it was resistant to Tulcea 50%,
but it was slightly sensitive (8 mm) to Arge 50%
solution.

7.

Conclusions

9.

8.

The best antibacterial activity on the bacterial

cultures tested, was registered for the 50%
concentrated solution from Tulcea area.
The Pseudomonas sp. strain was sensitive only to
50% concentrated aqueous solution obtained in the
Arge area.
The Candida albicans and Enterococcus sp. strains
have proven to be resistant to both concentrations
of the two tested solutions.
Our study results show that the two aqueous
extracts from Prunus spinosa L. species obtained
from the two different geographical areas could be
incorporated in some pharmaceutical formulas for
obtaining antibacterial effects.

10.

11.

12.

13.

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