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Phytochemical screening, antioxidant and antimicrobial activities of Algerian Cistus salvifolius extracts.

INTRODUCTION

Humans body are continuously exposed to free radicals created by various endogenous natural sources or environmental aggressor, which lead to an increase in the production of reactive species, causing cellular damage with adverse effects on lipids, proteins and DNA. Free radicals are involved in many human diseases and, in long term, contribute to aging, heart disease, atherosclerosis, diabetes, cancer and other many chronic diseases [1]. Moreover, reactive oxygen species (ROS) including free radicals and non free radicals are the main cause of deterioration of many foods, which lead to the formation of toxic compounds and minimize the nutritional value of foods. On the other hand, there are new concerns about food safety due to increasing occurrence of new food-borne disease outbreaks caused by pathogenic microorganisms. Many researchers reported the role of bioactive compounds from natural sources that may be used for human consumption sources to prevent against diseases (2), reduce oxidative stress or/and have antimicrobial activity [3, 4]. Antioxidants can exert their protecting effects by scavenging free radicals or preventing the generation of ROS, a subsequently, they retard the progress of many chronic diseases. Furthermore, antioxidants from natural sources can also increase the shelf life of foods.

Due to the increase of resistance to antibiotics, there is a pressing need to develop new and innovative antimicrobial agents. Currently, there is growing interest to use plant extracts for treatment of infectious disease and for the preservation of foods [5] as these possess a characteristic flavor and sometimes show antioxidant activity as well as antibacterial activity [6, 7, 8].

Cistaceae is a Mediterranean family of almost 200 species of shrubs. Some of these plants are wide spread in the north western Africa [9, 10]. Cistus species are used as an antidiarrheics, as general remedies in folk medicine for treatment of various skin diseases and as anti-inflammatory agents [11]. It has been reported that these plants exhibit a variety of biological activities such as antispasmodic [12], antialgal, antibacterical, antifungal, antiprotozoal, enzyme inducing, modulation of immune cell functions [13, 14], as well as cytotoxic effects against human cancer cell lines [15]. Among these plants, Cistus salviifolius L. (C. salvifolius) has been used against bronchitis as an expectorant and to stop bleeding [16]. Phytochemical reports on C. salvifolius concern the isolation of several phenols, including flavan-3-ols, oligomeric flavan-3-ols, oligomeric anthocianidins, protodelphinins, ellagitannins and the glucoside phenylpropanoid rhododendrin (betuloside) with analgesic and anti-inflammatory properties [17, 18]. Due to the ethnobotanical importance of the Cistus species and the fewer studies that prove their antioxidant and antimicrobial activities, the aim of this study was to determine the total phenolic content of the methanol and aqueous extracts of Cistus salvifolius and evaluate its antioxidant and antibacterial activities by using a series of in vitro tests.

MATERIALS AND METHODES

Chemicals:

Gallic acid, quercetin, ferrozine [3-(2-pyridyl)-5, 6-bis (4-phenylsulfonicacid)-1,2,4-triazine)], iron(II) chloride (Fecl2), butylated hydroxytoluene (BHT) were purchased from Sigma (Germany). 1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Fluka (Germany). All other reagents were from Sigma and Fluka (Germany) and were of analytical grade.

Plant material:

Cistus salvifolius was collected in May 2010 from Setif area, Algeria. The plant was identified and, authenticated taxonomically by Dr. N. Boulaacheb, Univesity of Setif 1, Algeria. A voucher specimen (No. C.S. 2010-2) was preserved at the local Herbarium of Botany, Department of Botany, University of Setif 1 for future reference. The leaves were shadow-dried and pulverized to dry powder.

Microorganism strains:

Gram-negative bacterial strains (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 13311, Acinetobacter baumanii ATCC 19606, Proteus mirabilis ATCC 35659, Klebsiella pneumoniae ATCC 700603) and Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC 49452, Lysteria monocytogenes ATCC 15313) were provided from American Type Culture Collection (ATCC).

Preparation of Cistus salvifolius extracts:

Methanol extract (Met E) was prepared by maceration of 100 g of powdered plant material with 80% methanol (1:10) at room temperature for 48h with frequent agitation. After filtration, the filtrate was concentrated by a rotary evaporator at 40[degrees]C and the residue was lyophilized to give a brown powder (yield: 23.27%).

Aqueous extract (Aq E) was prepared according to the traditional method by boiling 50 g of powdered plant in 500 ml of distillated water for 20 min. After filtration, the filtrate was lyophilized to give a brown powder (yield: 9.45%).

Polyhenol, flavonoid and tannin determination:

Total polyphenolic content was determined using the Folin-Ciocalteu [19]. Aliquots of test samples (100 [micro]l, 1mg/ml) were mixed with 0.4 ml, of 20% [Na.sub.2]C[O.sub.3] and incubated at room temperature for 4 min. After the addition of 500 [micro]l Folin-Ciocalteau's phenol reagent 10% in water, the reaction tubes were further incubated for 2 h at room temperature and finally the absorbance was read at 760 nm. Gallic acid was used as a standard. The concentration of total phenolic compounds in methanol and aqueous extracts was determined as mg of gallic acid equivalents per g of extract (GAE/g extract).

Total flavonoid content was quantified according to Bahorun et al. [20]. Briefly, samples of 1ml were incubated in the presence of 1ml of Al[Cl.sub.3] (2%), then the colored product of the reaction was quantified by measuring absorbance at 430 nm. Quercetin was used as a standard. Total flavonoid content was expressed as mg quercetin equivalent per g of extract (QAE/g extract).

Tannin content was determined using the hemoglobin precipitation assay according to Bate-Smith [18]. An aliquot of 0.5 ml of each extracts is mixed with 0.5ml of hemolysis bovine blood to reach a final concentration of 1mg/ml, then the mixture was centrifuged at 480g for 20 min and the absorbance was measured at 578 nm. Tannic acid was used as a standard and tannin content was expressed as mg tannic acid equivalentper g of extract (TAE/g extract).

HPLC-TOF/MS analysis:

HPLC-TOF/MS analysis was carried out as described elsewhere [22]. This HPLC-TOF/MS was developed to analyse phenolic acids and flavonoids in the extracts using Agilent Technology of 1260 Infinity HPLC System coupled with 6210 Time of Flight (TOF) LC/MS detector and ZORBAX SB-C18 (4.6 x 100mm, 3.5gm) column. The mobile phase consisted of solvent mixtures ultra-pure water with 0.1% formic acid and acetonitrile, respectively. Flow rate was 0.6 ml [min.sup.-1] and column temperature was 35[degrees]C. The injection volume was 10 [micro]l, and the solvent program was as follow: 0-1 min 10% B; 1-20 min 50% B; 20-23 min 80% B; 23-25 min 10% B; 25-30 min 10% B. Ionization mode of HPLC-TOF/MS instrument was negative and operated with a nitrogen gas temperature of 325[degrees]C, nitrogen gas flow of 10.0 L [min.sup.-1], nebulizer of 40 psi, capillary voltage of 3500 V and finally, fragmentor voltage of 175 V. For sample analysis, dried crude extracts (200 ppm) were dissolved in methanol at room temperature. Samples were filtered passing through a PVDF (0.45 [micro]m) filter by an injector to remove particulates.

DPPH radical scavenging assay:

The stable radical DPPH was used to measure the free radical scavenging activity by the method of Que et al. [23]. The solution of the free DPPH radical in ethanol (0.1 mM) was prepared and 1 ml of this solution was added to 1ml of plant methanol and aqueous extracts samples and standard (BHT) at various concentrations (2100 [micro]g/ml). Negative control contained 1ml of ethanol and 1ml of DPPH solution. All the reaction mixtures were incubated for 30 min at room temperature. The scavenging activity of DPPH radical by the plant samples was measured at 517 nm against methanol.

Reducing capacity determination:

The reducing power was determined according to the method of Oyaizu [24]. Each extract (0.02-0.35 mg/ml) in 2.5 ml of distillated water was mixed with 2.5 ml of 200 mM sodium phosphate buffer (pH 6.6) and 2.5 ml of 1% potassium ferricyanide and the mixture was incubated at 50[degrees]C for 20 min. Then, 2.5 ml of 10% trichloroacetic acid were added and the mixture was centrifuged at 200xg for 10 min. The upper layer (2.5 ml) was mixed with 2.5 ml of deionized water and 0.5 ml of 0.1% ferric chloride. Finally the absorbance was measured at 700 nm against a blank. BHT was used as a standard antioxidant.

Determination of total antioxidant activity:

The total antioxidant activity of methanol and aqueous extracts of C. salvifolius was determined according to Gulcin et al. [25] with slight modifications. Linoleic acid emulsion were prepared with 0.028 g of tween-20 and 0.028g of linoleic acid and 10 ml of 0.04M potassium phosphate buffer (pH 7). The solutions which contained 600 [micro]l of extracts or BHT (50 [micro]g/ml) was mixed to 600 [micro]l of linoleic acid emulsion. On the other hand, control was composed of 600 [micro]l linoleic acid emulsion and 600 [micro]l of 0.04M potassium phosphate buffer (pH 7). The mixed solutions were incubated at 25[degrees]C in the dark. The peroxide level was determined after reaction of 20 [micro]l of solutions with 20 [micro]l of fe[Cl.sub.2] (in 3.5% HCl) and 20 [micro]l of Thiocyanate. The absorbance of each mixture was determined at 500 nm after 15 min of reaction and every 24 h for 96 h. High absorbance indicates a high linoleic acid emulsion oxidation and inhibition of lipid peroxidation in percent was expressed as antioxidant capacity.

Determination of ferrous ions chelating:

The chelating of ferrous ions by the methanol and aqueous extracts of Cistus salvifolius was estimated by the method of Li et al. [19]. Briefly, the extracts samples (50-250 mg/ml) were added to a solution of 0.6 mmol/l Fe[Cl.sub.2] (50 [micro]l). The reaction was initiated by the addition of 50 [micro]l of ferrozine (5 mM) and the mixture was shaken vigorously and left standing at room temperature for 10 min. Absorbance of the solution was then measured at 562 nm. The percentage of inhibition of ferrozine-[Fe.sup.2+] complex formation was calculated. EDTA was used as a reference.

Screening for antibacterial activity by disc diffusion method:

The antibacterial activity was evaluated using the disc diffusion method against Gram-negative bacteria (Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 13311, Acinetobacter baumanii ATCC 19606, Proteus mirabilis ATCC 35659, Klebsiella pneumoniae ATCC 700603) and Gram-positive bacteria (Staphylococcus aureus ATCC 25923, Bacillus cereus ATCC 10876, Enterococcus faecalis ATCC 49452, Lysteria monocytogenes ATCC 15313). Suspensions of each specie was cultivated overnight at 37[degrees]C in a liquid medium nutrient. After that, the concentration of each species was normalized in isotonic sodium chloride solution for an optic density of 0.5 Mac Farlend (corresponding approximately to 1. [10.sup.6] cells/ml) by absorbance determination at 600 nm. Petri dishes of sterile Mueller-Hinton agar were seeded with the appropriate bacterial suspension. Sterile 6 mm diameter filter paper disc were impregnated with aqueous and methanol extracts immediately in volumes of 10 [micro]l corresponding to 200 and 300 [micro]g/ml. Amoxicilin (30 [micro]g/disc) was included in the test as a reference antibiotic control. After incubation at 37[degrees]C for 24 h the inhibition zones of growth were recorded in mm [26]. Antibacterial activity was expressed as a ratio of the inhibition zones caused by the plant extract and amoxicilin. The solvent used was tested and did not show any inhibition on bacterial growth. The results are averages of triplicate tests.

Statistical analysis:

The experimental results were expressed as means [+ or -] SD of triplicate values. The results were subjected to one way analysis of variance (ANOVA) and the significance of differences between samples means were calculated by Graph pad prism 5.0 using Tukey multiple range test. P values [less than or equal to] 0.05 were regarded as significant.

Results:

Polyphenol, flavonoid and tannin contents:

The obtained values of total phenolic, flavonoid and tannin contents of aqueous and methanol extracts of Cictus salvifolius are presented in Table 1. Methanol extract contains the highest amount of polyphenols, flavonoids and tannins. The amounts of polyphenols and tannins in the aqueous extract are very close.

HPLC-TOF/MS analysis:

Results of HPLC-TOF/MS analysis revealed the presence of phenolic acids and flavonoids in C. salvifolius methanol and aqueous extracts (table 2). The conditions used led to a good separation of the peaks which could be identified in the chromatogram (figure 1A and B). Both extracts of C. salvifolius contain the highest amount of gallic acid with retention time (RT) = 2.051 and gentisic acid, followed by protocatechuic acid. The 4hydroxy benzoic acid, cinnamic acid and furillic acid were only detected in methanol extract. Among the tested flavonoids, catechine and rutin were detected in both extracts but the methanol extract contains the highest amount of these flavonoids. Apigenin-7-glucoside was only detected in the methanol extract.

DPPH radical scavenging activity:

At 20 [micro]g/ml, methanol and aqueous extracts of C. salvifolius showed a significant free radical scavenging activity with 92.02% and 82.89%, respectively (figure 2). The best free radical scavenging activity was exerted by methanol extract with [IC.sub.50] = 6.79 [micro]g/ml flowed by aqueous extract with [IC.sub.50] = 9.37 [micro]g/ml. These values are better than that obtained with BHT, used as standard antioxidant.

Reducing power:

The reducing power of C. salvifolius extracts was concentration-dependant manner (figure 3). Methanol extract has the stronger reducing power with [IC.sub.50]=5.29 [micro]g/ml than the aqueous extract ([IC.sub.50]=11.64 [micro]g/ml). However, the reducing power of bHt used as reference was relatively less pronounced ([IC.sub.50]=16.99 [micro]g/ml).

Metal chelating activity:

Methanol and aqueous extracts of C. salvifolius exhibited a weak metal chelating activity; and this effect is concentration-dependent manner (figure 4). Indeed, at 100 [micro]g/ml, methanol and aqueous extracts exerted only 17.66 % and 24.32% chelation of ferrous ion, respectively. However, the chelation augmented at the highest concentrations, and the [IC.sub.50] for methanol extract, aqueous extract and EDTA were 321.70 [micro]g/ml, 644.15 [micro]g/ml and 5.97 [micro]g/ml, respectively. The difference among all concentrations of the two extracts and the reference EDTA was statistically significant (P<0.001).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

[FIGURE 3 OMITTED]

Total antioxidant activity:

The total antioxidant activity of methanol extract, aqueous extract and BHT is shown in figure 5. High absorbance indicates a high linoleic acid emulsion oxidation. At the same concentration, both extracts and BHT exerted similar antioxidant effect on lipid peroxidation in linoleic acid system. The percentages of inhibition are 79.19%, 76.02%, and 77.39%, respectively. The inhibitory effect was maximal at 24h and remained steady until 96h.

[FIGURE 4 OMITTED]

[FIGURE 5 OMITTED]

Evaluation of antibacterial activity:

The antibacterial effect of aqueous and methanol extracts of C. salvifolius against Gram-positive and Gram-negative bacteria is shown in table 3. In the Gram-positive bacteria, both extracts at the used concentrations (200 [micro]g/ml and 300 [micro]g/ml) presented antimicrobial activity against Bacillus cereus with zones of inhibition between 11.66 and 16.55 mm, Enterococcus faecalis with zones of inhibition between 12.66 and 16.88 mm and Lysteria monocytogenes with zones of inhibition between 10.22 and 12.33 mm. No effect was observed against Staphylococcus aureus. The most pronounced effect was observed against Gram-negative bacteria in the order: Salmonella typhimurium > Proteus mirabilis > Acinetobacter baumanii > Pseudomonas aeruginos > Escherichia coli. No effect was observed against Klebsiella pneumoniae. In general, aqueous and methanol extracts showed similar antibacterial effect, except against Salmonella typhimurium and Acinetobacter baumanii where aqueous extract showed the lowest activity. Some variability was observed in amoxicilin activity against the tested bacteria, which could be explained by less sensibility of some strains to this antibiotic.

Discussion:

Antioxidants from natural sources can replace the synthetic antioxidants which are restricted for their toxic and carcinogenesis effects. These bioactive compounds from plants may be used for human consumption to prevent or reduce oxidative stress or/and have antimicrobial activity [3, 4]. In the present study, the methanol and aqueous extracts of Cistus salvifolius were screened for the phytochemical constituents, and then examined for the antioxidant and antimicrobial activities in vitro.

The high quantity of polyphenols, tannins and flavonoids was observed in the methanol extracts of C. salvifolius leaves. Phenolic compounds are generally more soluble in organic solvents than in water, which may explain the difference in the phenolic contents between the two extracts. The HPLC-TOF/MS revealed the presence of flavonoids and phenolic acids such as catechin, rutin and galic acid as major constituents. Some compounds which are present in the methanol extract in a small quantities are absent in the aqueous extract. This result is supported by the study of Toniolo et al. [27] on C. salvifoliu, whih revealed chemical differences in the plant extracts. As reported in the previous studies, the presence of these compounds is known to support the bioactivities of medicinal plants. Indeed, it has been reported that the antioxidant activity of plant origin components can be attributed mainly to the presence of phenolic compounds [28]. These compounds are the main class of natural antioxidants and there is a close relationship and positive correlation between the phenolic content and antioxidant activity of plant extracts [29].

Antiradical activity of methanol and aqueous extracts from C. salvifolius was studied by screening their possibility to bleach DPPH radical. This stable free radical accepts an electron or hydrogen radical to become a stable diamagnetic molecule which is widely used to investigate radical scavenging activity. The DPPH radical scavenging activity of the both extracts is concentration-dependent. At the same concentration (20[micro]g/ml), methanol extract which showed the highest content of phenolic compounds exhibited the highest scavenging activity (92.02%). The antioxidant activity of phenolic compounds is mainly due to their redox properties, which can play an important role in absorbing and neutralizing free radicals by their hydrogen donating ability [30], quenching singlet and triplet oxygen or decreasing peroxides [31].

The stronger reducing power observed in the methanol extract than in the aqueous extract was due to the presence of reductones and may serve as an indicator of its potent antioxidant activity. The antioxidant effect of reductones is based on the destruction of the free radical chain by donating a hydrogen atom [30]. The reduction of ferrous ion ([Fe.sup.3+]) to ferric ion ([Fe.sup.2+]) is measured by the strength of the green-blue color of solution which absorbs at 700 nm. Polyphenols which may act in a similar way as reductones react with free radicals to turns them into more stable products and abort free radical chain reactions [32].

The metal chelating activity is based on chelating of [Fe.sup.2+] ions by reagent ferrozine, which lead to the formation of a complex with [Fe.sup.2+] ions [33]. The formation of this complex is probably disturbed by other chelating reagents, which would result in the reduction of the formation of red-colored complex. In this study, both extracts interfered weakly with the formation of ferrous complex with the reagent ferrozine, suggesting the the weak chelating activity of the extracts and the captures of the ferrous ion before ferrozine. Metal chelating capacity of both extract was significant, since it reduced the concentration of the catalysing transition metal in lipid peroxidation system. It has been reported that chelating agents are effective as secondary antioxidants as they reduce the redox potential, thereby stabilizing the oxidized form of the metal ion [34].

Lipid peroxidation leads to rapid development of rancid and stale flavours and is considered as a primary mechanism of quality deterioration in lipid foods and oils. During the linoleic acid oxidation, peroxides are formed, which oxidize [Fe.sup.+2] to [Fe.sup.+3]. The latter ion forms a complex with thiocyanate and this complex has a maximum absorbance at 500 nm. Results showed that methanol and aqueous extracts of Cistus salivifolius inhibited the peroxidation of linoleic acid. Lipid peroxidation is thought to proceed by radical mediated abstraction of the hydrogen atom from a methylene carbon in a polyunsaturated fatty acid side chain [35], and the inhibitory effects on lipid peroxidation and autoxidation of linoleic acid have been attributed to the radical scavenging activity [36]. This ability to modify the induced lipid peroxidation by free radicals is linked not only to the structural characteristics of the antioxidants agents but also to their ability to interact with and penetrate the lipid bilayer [37]. It has been shown that the structure and the lipophilicity of polyphenols are determinant factors of antioxidant properties of these compounds in the lipid layer of the membrane [38].

All the antioxidant effects of Cistus salvifolius observed in this study could be due to the presence of flavonoids and phenolic compounds which are considered to be free radical scavengers and chain-breaking antioxidants [39].

On the other hand, plant extracts are potential sources of novel antimicrobial compounds (40=40), especially against pathogen bacteria. These bacteria are the main factors for the pathogenesis of various diseases as well as for the deterioration and spoilage of pharmaceutical, cosmetic and food products. In the last decade, due to the increase of resistance to antibiotics, there is a pressing need to develop new and innovative antimicrobial agents (46=41). According to the results of this study, both extracts of C. salvifolius inhibited the growth of the majority of the bacteria tested. All the Gram-negative microorganisms (Pseudomonas aeruginosa, Escherichia coli, Salmonella typhimurium, Acinetobacter baumanii, Proteus mirabilis) and Gram-positive (Bacillus cereus, Enterococcus faecalis, Lysteria monocytogenes) were most susceptible to the two extracts, whereas the Gram-positive microorganisms Staphylococcus aureus and the Gram-negative Klebsiella pneumoniae were the most resistant. The methanol extract is the most effective and it also remarked that both extracts of Cistus salvifolius were, in some cases, more effective than the antibiotic amoxicillin used as reference, which suggests potential applications for these extracts as antimicrobial agents. The antimicrobial potential of plant extracts is widely studied [6, 7, 43=42]. The antibacterial activity exerted by both extracts might be attributed to the presence of bioactive constituents such as catechin, rutin and apigenin, gallic acid, gentisic acid cinnamic acids detected by HPLC-TOF/MS. Indeed, several studies reported the antimicrobial activity of polyphenols, tanins and flavonoides [43, 44]. Moreover, Haouat et al. [45] showed that the molecules responsible for the antimycobacterial activity of Cistus salvifolius and Cistus albidus are polyphenols.

Conclusion:

Cistus salvifolius extracts are rich in phenolic compounds and effective as antioxidants and antimicrobials. The use of these extracts as additives in foods could provide considerable benefits for the treatment of diseases related to reactive species production and oxidative stress and against bacterial infections, or in the food industry as they could retard oxidative degradation of lipids and thereby improve the quality and nutritive value of food. So, Cistus salvifolius can be considered as potent natural antioxidant and antimicrobial source for medicinal, cosmetics and food applications.

Received 28 December 2015; Accepted 28 January 2016; Available online 24 February 2016

ACKNOWLEDGEMENTS

This study was supported by grants from the Algerian Ministry of High Education and Cankiri Karatekin University.

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(1) Seoussen Kada, * (1) Hamama Bouriche, Abderrahmane Senator, (2) Fatih Gul and (2) Ibrahim demirtas

(1) Laboratory of Applied Biochemistry, Faculty of SNV, University Ferhat Abbas, Setif 1, Algeria

(2) Department of Chemistry, Faculty of Science, Cankiri Karatekin University, Cankiri, Turkey

Address For Correspondence:

Hamama Bouriche, Laboratory of Applied Biochemistry, Faculty of SNV, University Ferhat Abbas, Setif 1, Setif 1900, Algeria.

E-mail: bouriche_ha@yahoo.fr
Table 1: Polyhenols, tannin and flavonoid contents in
methanol extract (Met E) and aqueous extract (Aq E)
of Cistus salvifolius.

            Polyphenols              Tannins
         (mg GAE/g extract)     (mg TAE/g extract)

Met E   414.71 [+ or -] 0.01   374.56 [+ or -] 0.01
Aq E    291.50 [+ or -] 0.08   278.31 [+ or -] 0.01

            Flavonoids
         (mg QE/g extract)

Met E   14.13 [+ or -] 0.03
Aq E    4.86 [+ or -] 0.04

Values are mean of triplicate determination (n = 3)
[+ or -] SD.

Table 2: Phenolic acids and flavonoids in methanol and
aqueous extracts of Cistus salvifolius (CS) determined
by HPLC-TOF/MS.

                 Methanol extract            Aqueous
                                             extract

Coumpounds         RT     Concentration   Concentration
                            mg/kg CS        mg/kg CS
Gallic acid      2.051       122.26          383.20
Gentisic acid    4.386        45.46           18.03
Catechin         6.139         899           143.15
4-hydroxy        6.123        3.882            --
  benzoic acid
Protocatechuic   6.982        19.96           9.869
  acid
Rutin            9.496       211.253          45.95
Ferullic acid    11.164       9.121            --
Apigenin-7-      12.303      11.697            --
  glucoside
Cinnamic acid    15.703      66.526            --

Table 3: Antibacterial activity of Cistus salvifolius
methanol and aqueous extracts against Gram-positive and
Gram-negative bacteria.

                         Methanol extract

                              200gg/ml              300gg/ml

Bacteria strains         Zone of inhibtion (mm)

Pseudomonas aeruginosa   12.44 [+ or -] 0.19   14.21 [+ or -] 0.5
Escherichia coli         10.33 [+ or -] 0.33   11.55 [+ or -] 0.19
Salmonella typhimurium   27.44 [+ or -] 0.5    27.22 [+ or -] 0.19
Acinetobacter baumanii   15.44 [+ or -] 0.19   15.44 [+ or -] 0.50
Proteus mirabilis        20.22 [+ or -] 0.19   20.88 [+ or -] 0.19
Klebsiella pneumoniae            --                    --
Staphylococcus aureus            --                    --
Bacillus cereus          13.22 [+ or -] 1.26   16.55 [+ or -] 0.19
Enterococcus faecalis    12.66 [+ or -] 0.57   12.77 [+ or -] 0.38
Lysteria monocytogenes   10.22 [+ or -] 0.19   10.33 [+ or -] 0.33

                         Aqueous extract

                              200gg/ml               300gg/ml

Bacteria strains         Zone of inhibtion (mm)

Pseudomonas aeruginosa   10.44 [+ or -] 0.5    12.00 [+ or -] 0.05
Escherichia coli         14.88 [+ or -] 0.19   15.22 [+ or -] 0.19
Salmonella typhimurium   14.55 [+ or -] 0.38   14.55 [+ or -] 0. 69
Acinetobacter baumanii    10.33[+ or -]0.33    11.11 [+ or -] 0.19
Proteus mirabilis        19.55 [+ or -]0.19    20.44 [+ or -] 0.19
Klebsiella pneumoniae            --                     --
Staphylococcus aureus            --                     --
Bacillus cereus          11.66 [+ or -] 1.15   12.11 [+ or -] 0.19
Enterococcus faecalis    15.55 [+ or -] 0.50   16.88 [+ or -] 0.19
Lysteria monocytogenes   11.33 [+ or -] 0.57   12.33 [+ or -] 0.33

                               Amoxicilin

                               30gg/disc

Bacteria strains         Zone of inhibtion (mm)

Pseudomonas aeruginosa             --
Escherichia coli                   --
Salmonella typhimurium    19.66 [+ or -] 0.58
Acinetobacter baumanii             --
Proteus mirabilis         10.32 [+ or -] 0.58
Klebsiella pneumoniae              --
Staphylococcus aureus              --
Bacillus cereus                    --
Enterococcus faecalis      12.0 [+ or -] 0.0
Lysteria monocytogenes             --

(--) absence of susceptibility
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Author:Kada, Seoussen; Bouriche, Hamama; Senator, Abderrahmane; Gul, Fatih; demirtas, Ibrahim
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:6ALGE
Date:Jan 1, 2016
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