Antibacterial activity of two extracts from Rubus fruticosus L . against resistant pathogens and their antioxidant potential

1 Laboratory of Applied Microbiology in Food, Biomedical and Environment (LAMAABE), Department of Biology, Faculty of Nature and Life, Earth and Universe Sciences, Aboubekr Belkaid University of Tlemcen, 13000 Tlemcen, Algeria. 2 Laboratory of Valorization and Conservation of Biological Resources (VALCOR), Department of Biology, Faculty of Sciences, University of Boumerdes, 35000 Boumerdes, Algeria.


INTRODUCTION
Respiratory infections are considered as a major risk for human health, beside emerging respiratory infections such as severe acute respiratory syndrome (SARS), avian influenza and Hantavirus pulmonary syndrome, *Corresponding author.E-mail: afifchaouchethanina@yahoo.fr.Tel: 00 213 771 79 44 49.
Author(s) agree that this article remains permanently open access under the terms of the Creative Commons Attribution License 4.0 International License some respiratory infections are considered as eradicated or nearly, reappear with serious consequences: every year more than 8 million cases of severe respiratory infections are reported causing 3.9 million death (Sansonetti and Orth, 2006).
Despite the fact that researchers developed an important arsenal to face these infections and others, emerging resistances to antibiotics come to complicate the situation, this resistance implies various mechanisms such as modifying or synthesizing new targets, enzymatic modification, increasing efflux of antibiotics or others, the result is an under activity or non-activity of an important number of antibiotics used nowadays.
Many solutions were suggested to face this situation, and one of the most promising options is trying to extract from the plants new molecules that can be safe and effective in healing such diseases, the number of medicinal plants are estimated to be between 40000 and 70000 which are very diverse source of bioactive molecules and about 80% of world population is currently using these plants (Ramawat, 2009).
The interest in using these plants by population and also in research and academic world is increasing; new journals and reference books are published to enable researchers to find evidence based knowledge.Rubus fruticosus L. (Rosaceae) is a shrub well known for its fruit, called blackberries which are marketed all over the world for its delicious taste and high nutritive value.The shrub may be original from Armenia and is nowadays spreading in Europe, Asia, Oceania, northern and southern America and northern Africa (Hummer and Janick, 2007;Swanston-Flatt et al., 1990;Zia-Ul-Haq et al., 2014).
This plant is understory vegetation, can reach 3 m in length and have spiny stems.It flourishes at the end of spring and the fruit ripens in autumn (Hummer and Janick, 2007).According to some authors, this plant can be a good remedy for bronchitis and respiratory infections (Blumenthal et al., 1998).
However, no studies are reported on the Algerian R. fruticosus L. Two studies in Iran and Pakistan were reported by the antimicrobial activity of this species, two others were interested with the antioxidant activity.
The purpose of this study was to highlight the antimicrobial activity of this plant on both reference strains and strains isolated from patients with respiratory infections and in the same time, the study shows the antioxidant properties of its flavonoids extract and essential oil.

Biological material
The harvest of the aerial part of R. fruticosus was accomplished in spring in the area of Tizi Ouzou, in the north east of Algeria, about 80 km of Algiers, the capital, between 36° 43′ 00″ North and 4° 03′ 00″ East.To extract flavonoids, collected parts were dried in open air and sheltered from light, then transformed into powder using an electrical crusher; this powder was maintained in tightly closed glass flasks.
The tested strains was either reference strains or isolated from patients diagnosed with respiratory infections and hospitalized in the department of infectious diseases in the hospital university of Tizi Ouzou, the identification was effected using biochemical galleries Api 20E, Api 20NE, or specific tests.

Extractions
The extraction of essential oil was effected by hydro distillation, to accomplish this, 100 g of fresh matter was soaked in a recipient of 1l filled with 600 ml of distilled water, the whole system was boiled for 3 h.Collected vapors were condensed by transiting a refrigerator and collected.Organic state was recovered by adding few milliliters of diethyl ether and the obtained oil was kept in a temperature between 0 and 4°C.
The extraction of flavonoids was realized according to Bruneton (1999) protocol.This extraction is based on the difference of solubility degrees of flavonoids in various organic solvents.This protocol includes two main steps: first, methanol is used to solubilize flavonoids and then washings using petroleum ether, diethyl ether, ethyl acetate and butanol are realized.After using butanol, the extract contains the most polar flavonoids.

GC-MS of essential oils
Analysis of the chemical composition of the essential oil was carried out by chromatography and gas chromatography coupled with mass spectrometry, the apparatus was a GC Perkin Elmer 600, MS Perkin Elmer 600C, a column Rtx-VMS menu (60m long with a diameter of 250 µm).The carrier gas was helium with a flow rate of 1ml/min.0.2 μl oil was used to analyze injection using a special syringe.
The temperature was 70°C for 1 min, ramp 3to 160°C, 1 min ramp 2°C/min to 230°C for 5 min.The injector temperature was 230°C.

Dosage of flavonoids using a spectrophotometry method
To dose flavonoids, we used the aluminum chloride colorimetric method (Bahorun et al., 1996).Absorbance was read by a spectrophotometer (Optizen 2120 UV) at 430 nm.To determine the concentration of flavonoids in the extract, a calibration range was established using quercetin (1-25 µg/ml).The results of dosage are expressed in equivalent micrograms of quercetin for each gram of the extract.

High performance liquid chromatography
HPLC was used to achieve the quantitative analysis of flavonoids.
Column of silica was used as stationary phase (C18 reverse phase), this column measures 125 by 4.6 mm.A mixture of water/methanol/acetic acid (50:47:2.5)was used as a mobile phase, in isocratic system with a flow of 1 ml/min (Amarowicz et al., 2005).Extracts and standards were both analyzed with concentration of 0.5 mg/ml.The used volume was 20 μl.Detection was achieved by a UV-Visible detector at 254 nm.

Antimicrobial activity
A steers machine which is a multiple seeding instrument was used to facilitate the study of the antimicrobial activity of essential oil and this is according to the recommendations of the French Society of Microbiology.After diluting essential oil in mediums: Mueller Hinton for bacteria or Sabouraud for fungi, they were let to solidify.Bacterial suspensions of 0.5 Mc Farland was put using spots.For assessing the flavonoic extract activity, plates of Mueller Hinton agar for bacteria and Sabouraud agar for fungi were inoculated by swabbing of standardized microbial suspension (0.5 Mc Farland), according to NCCLS recommendations (NCCLS, 2006), after that, on the agar, we placed discs of 6 mm diameter containing 10 μL of extract with different concentrations.
As positive control, discs containing antibiotics was used and placed in the center of the plate.After incubating for 24 h at 37°C for bacteria and 48 h for fungi, we determined the antimicrobial activity of both extracts by measuring the MICs.Every test was performed in triplicate.

The DPPH test
To quantify the antioxidant activity of both flavonoids extract and essential oil, we used the method of Sanchez Moreno et al. (1998).
For that, we prepared various concentrations of 0.1; 0.2; 0.4; 0.6; 0.8 and 1 mg/ml using a stock solution of both extracts obtained by dissolution in methanol.1 ml from each one of the concentrations was added to 4 ml of DPPH solution whose concentration was 0.024 mg/ml, this was the preparation for assessing the activity of essential oil.For flavonoids, we mixed 25 µl of each concentration with 975 µl of the same solution of DPPH.Ascorbic acid was prepared using the same protocol.After 30 min, we measured the variation of absorbance using UV-visible spectrophotometer (Optizen 2120 UV), driven by a computing system in the wavelength of 517 nm.To express results in percentage, we used this formula: A reference is the absorbance of the control which contains only reactive.A Test is the absorbance of the extract.

Statistical analysis of the data
Results of the antioxidant activity were expressed as mean ± SD.

Phytochemical screening
The phytochemical screening of R. fructicosus L. reveals abundant amount of total tannins, catechin tannins, gallic  tannins, flavonoids, senosids, saponosids, coumarins (Table 1).This plant seems to have a high potential but there is no sufficient studies on its chemical composition and biological activities.
A study on the same species shows that it contains alkaloids, flavonoids, tannins, saponins and glycosides (Rameshwar et al., 2014).We found similar results with no glycosides, but the presence of other compounds such as sénosides and coumarins.

Extractions
Using 100 g of dried matter, the yield was 0.05 ± 0.01% of essential oil.We obtained lightly viscous oil, with a pale yellow coloration and a characteristic odor.The aqueous extract has a gelatinous aspect and brownish color.The yields of extraction using different solvents (diethyl ether, ethyl acetate, butanolic and aqueous) vary between 2.99 and 63.2% (Table 2).
Blackberry has about 30% of highly polar flavonoids (weak) against more than 60% butanol extract (the most abundant extract) in the plant of the present study.Total  polyphenols of one Rosaceae plant contain a yield of about 140 mg/100 g of the material, although this performance is quite high, but we cannot show the percentage of each phenolic extract (Benvenuti et al., 2006).

Dosage of flavonoids
Content of flavonoids is expressed in equivalent mg of quercetin/ml of the extract of the whole plant.It was found to be 75.54mg/ml.The content of R. fruticosus in flavonoids cannot be discussed because there is no published work on the chemical composition of this plant or another plant belonging to the same family, but by observing the averages of yields of extraction in many studies, 75.54% is a high yield and we it can be assumed that this extract is high in flavonoids.

High performance liquid chromatography
On the chromatogram of flavonoids of R. fruticosus L., we found eight peaks corresponding to eight main compounds which are: gallic acid, gallocatechin, protocatechic acid, catechin, caffeic acid, rutin, ellagic acid and myricetin.Results are shown in Table 3 and Figure 1.An analysis by HPLC made in South America by Mertz et al. (2007) showed the presence of flavonol hexoside-malonates and hydroxycinnamic acids in R. fruticosus extracts.
A study which was realized by the same authors using HPLC with diode array reveals the presence of cafeic acid in two neighbors species which are Rubus glaucus Bent and Rubus adenotrichus Schlech, this two plants are not rich in this component, the same component was find using HPLC in a variety of R. fruticosus obtained from the region of Tizi Ouzou in Algeria, that shows the presence of cafeic acid in the botanical kind, Rubus.
Another study has revealed, using HPLC, the presence of ellagic acid and 3,4-dihydroxy-benzoic acid in the flavonoids issues from leaves of R. fruticosus gathered in Bulgaria.Ellagic acid was also detected in the aerial part of the studied plant.Other molecules were identified in this study and were never identified in previous studies,  this may be explained by the difference of the area of harvest (Milivojevic et al., 2011;Radovanović et al., 2013;Carlsen et al., 2003).

GC-MS of essential oil of R. fruticosus
By analyzing the essential oil of R. fruticosus using GC-MS, we identified 11 molecules (Figure 2).The major component is the lanceol (20.22%) (Table 4).Concerning the essential oil of R. fruticosus, no studies were done on it or any essential oil of any species belonging to Rubus family.

Antimicrobial activity
Table 5, the antimicrobial activity of flavonoids extract and the essential oil on reference strains are represented, both substances have an effect on the respiratory flora.The activity of both extracts was evaluated on strains isolated from patients with respiratory infections; these patients were diagnosed with respiratory infections, ORL infections or bronchitis infections.
Clinical strains chosen to evaluate antimicrobial activity showed an important resistance to conventional antibiotics, Table 6 shows the results.By assessing the antimicrobial activity of this species against some respiratory pathogens, this plant seems to be promising but no many works was done on evaluating the effect of the extract of this species against microbes.A study evaluated the activity of the ethanolic extract of R. fruticosus against Helicobacter pylori which was resistant to nalidixic acid, this extract had a MIC of 400-450 μg/ml (Abachi et al., 2013).
Another study showed that the blackberry juice inhibits the growth of Bacillus cereus, Bacillus subtilis, Streptococcus marcescens and Escherichia coli with percentages varying from 50 to 75% (Poyrazolu and Biyik, 2010).Flan et al. (2011) found that the methanolic extract of aerial parts of R. fruticosus inhibits the growth of Mycobacterium tuberculosis with a MIC of 1 mg/ml.Another study by Riaz et al. (2011) on methanolic extracts from various parts of the plant against eight reference bacterial strains (Salmonella typhi, E. coli, Streptococcus aureus, Micrococcus luteus, Proteus mirabilis, Bacillus subtilis, Citrobacteri sp., Pseudomonas aeruginosa), revealed that all the extracts inhibit the growth of bacteria with different MICs.All these studies showed that different extracts of R. fruticosus, especially phenols, have a big potential to inhibit the growth of bacteria or fungus.

DPPH test
Free radical scavenging was appreciated using DPPH discoloration test that revealed a high antioxidant activity of the flavonoic extract of R. fruticosus.This test revealed a weak scavenging of free radical activity for the essential oil, the EC 50 was 9.24 ± 0.48 mg/ml.This value is 2.68 ± 0.88mg/ml for flavonoids.In Figure 3, these results are compared with ascorbic acid as a standard.The antioxidant activity of flavonoids of R. fruticosus is closer to the values of reference which is ascorbic acid (IC 50 was found 1.46 ± 0.01mg/ml).
A study showed that IC50 varies from 15.2 to 76.5 µg/ml for different extracts including n-hexane, dichloromethane, chloroform, ethyl acetate, methanol.Blackberries are a rich source of natural antioxidants as they contain high levels of phenols and flavonols and are therefore well reputed scavengers and inhibitors of free radicals (Ivanovic et al., 2014).
The study of both biological activities: antimicrobial and antioxidant of the extract of flavonoids and essential oil of R. fruticosus reveals that the essential oil has a weak biological activity while flavonoids seem to have strong biological effects.

Conclusion
By assessing the antimicrobial and antioxidant activities of the flavonoic extract and the essential oil of Algerian R. fruticosus L., we can assume that this species and particularly its flavonoic extract have a remarkable inhibitory action against resistant respiratory pathogens.
More advanced studies are needed to investigate the possibility of developing formulations for pharmaceutical products from this extracts.

Figure 2 .
Figure 2. Chromatogram of GC-MS applied to essential oils.

Table 2 .
Yields in % of flavonoids extracts.

Table 3 .
Retention time of the used standards.

Table 4 .
Retention time of essential oil compounds.

Table 5 .
Results of the antimicrobial activity against reference strains.

Table 6 .
Activity of R. fruticosus extracts against respiratory pathogens.