Chemical analysis and biological activities of Populus nigra, flower buds extracts as source of propolis in Algeria

Industrial Crops and Products 53 (2014) 85–92

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Chemical analysis and biological activities of Populus nigra, flower buds extracts as source of propolis in Algeria Nadjet Debbache, Dina Atmani, Djebbar Atmani ∗ Laboratoire de Biochimie Appliquée, Universté Abderrahmane Mira, Bejaia 06000, Algeria

a r t i c l e

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Article history: Received 11 August 2013 Received in revised form 9 December 2013 Accepted 10 December 2013 Keywords: Populus nigra Antioxidant Antimicrobial Anti-inflammatory Reactive oxygen species Xanthine oxidase

a b s t r a c t Seven fractions obtained by a selective extraction procedure from Populus nigra flower buds were examined for their antimicrobial and antioxidant properties, in vitro, using radical scavenging and inhibition of lipid peroxidation assays. The aqueous chloroform fraction that exhibited the best antioxidant activity was investigated for anti-inflammatory activity in vivo. Quantification of total phenolic compounds in these fractions was equally carried out. The results obtained with the aqueous fraction of chloroform were particularly interesting, being the most effective on DPPH (IC50 = 24.61 ␮g/mL), ABTS (IC50 = 17.09 ␮g/mL), NO (IC50 = 9.52 ␮g/mL), HOCl (IC50 = 187.90 ␮g/mL) and OH− (IC50 = 113.79 ␮g/mL) radicals in addition to exerting a high inhibition on both xanthine oxidase (XO) activity and lipoperoxydation (IC50 = 60.7 and 24.93 ± 1.22 ␮g/mL, respectively). Moreover, the same fraction (200 mg/kg) has equally demonstrated a potent anti-inflammatory potential (62.36%) in carrageenan-induced mice paw edema model. On the other hand, it is rather the organic extracts that exhibited the highest antimicrobial activity against tested microorganisms. Hence, these results suggest that P. nigra is a promising source of bioactive compounds that can be exploited as antioxidants and bactericidal in food products as well as in pharmaceutical therapeutic use. © 2014 Published by Elsevier B.V.

1. Introduction In the wake of the propagation of diseases caused by oxidative stress such as cancer, Alzheimer and inflammatory related pathologies, it has become urgent to find natural non-toxic antioxidants devoid of adverse side effects. That is why researchers are accelerating the pace in the field of ethnopharmacology. Free radicals which induce oxidative stress are generated by several enzymes like xanthine oxidase involved in various inflammatory disorders (Granger et al., 1986). Moreover, their excessive formation by activated neutrophils implicates them in acute inflammation (Halliwell et al., 1988). Thus, antioxidants which prevent their formation are expected to suppress or at least attenuate the inflammatory reaction (Hazra et al., 2010). Anti-inflammatory drugs are reputed for their undesirable side effects. In this context, the quest for sources of novel antioxidants as substitutes is being pursued. Moreover, as the etiology of some inflammatory conditions is microbial, combating microorganisms is one way of eradicating the inflammatory response. The problem of microbial resistance to usual medications is growing which makes its use in the future uncertain, fueling the search for new, appropriate and efficient antimicrobial drugs of natural origin.

∗ Corresponding author. Tel.: +213 34214762. E-mail address: [email protected] (D. Atmani). 0926-6690/$ – see front matter © 2014 Published by Elsevier B.V. http://dx.doi.org/10.1016/j.indcrop.2013.12.018

It is well documented that in the temperate zone all over the world, the main source of bee glue is the resinous exudate of Populus nigra buds or propolis (Bankova et al., 1995). The biological activities of the latter, which have attracted both commercial and scientific interests, are attributed to the same plant-derived substances found in the original source (P. nigra) (Bankova, 2005). In addition, the use of these substances in food and food supplements is considered as safe by the United States Food and Drug Administration (FDA) and the Scientific Committee (SC) of European Food Safety Authority (EFSA) (EFSA Journal, 2012), meaning that they are devoid of toxic side effects characteristic of synthetic antioxidants. Algeria harbors a large variety of medicinal and aromatic plants which were proved valuable pertaining to their antioxidant properties (Djeridane et al., 2006; Atmani et al., 2009; Berboucha et al., 2010). Traditional uses of P. nigra flower buds in the treatment of many inflammatory-related conditions such as arthritis, bronchitis and respiratory tract diseases are wide. Previous investigations indicated that radical scavenging activity of plant extracts was dose- and radical/method specific (Hazra et al., 2010). Also, because of the complex nature of phytochemicals present in extracts and owing to the complexity of oxidation–antioxidation processes, antioxidant activities of plant extracts cannot be evaluated by a single method. To provide a comprehensive picture of the antioxidant nature of a given extract, commonly accepted assays, including enzymatic and nonenzymatic methods, were used to evaluate the total antioxidant

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effects of plant extract (Prabhakar et al., 2006; Parmar and Kar, 2009). Few data have been published on the biological activities of the buds extracts of P. nigra and the corresponding propolis. In this study, the antioxidant capacity of several extracts of P. nigra flower buds was investigated using several radicals, including hydrogen peroxide (H2 O2 ), hydroxyl (OH− ), nitric oxide (NO• ), hypochlorous acid (HOCl), 2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonate) (ABTS) and (2,2-diphenyl-1-picrylhydrazyl) DPPH. The aqueous fraction derived from chloroform which exhibited the best antioxidant potential was further investigated for anti-inflammatory activity in vivo. Anti-bacterial and anti-fungal activities of plant extracts were also assessed. Furthermore, total phenols, flavonoids and tannins in plant extracts were quantified.

hand. Obtained organic and aqueous fractions were dried to be used for different experiments. 2.6. Chemical analysis

2. Materials and methods

Total phenolic quantification of plant extracts was carried out using the Folin–Ciocalteu reagent and catechin as standard (Lowman and Box, 1983). The AlCl3 method described by Maksimoviˆc et al. (2004) was employed for the determination of flavonoid content of the sample extract. Concentrations of flavonoids were deduced from a standard curve and expressed as mg of quercetin equivalent/gram of extract. Tannins were determined by precipitation using the bovine serum albumin (BSA) method developed by Hagerman and Butler (1978). Concentrations of tannins were expressed as mg of tannic acid equivalent per gram of extract.

2.1. Chemicals

2.7. Antioxidant activity in vitro

All the reagents and chemicals were purchased from Sigma, represented by Prochima Sigma, Tlemcen, Algeria.

In all the tests, radical scavenging activity was calculated as follows: % radical scavenging activity = A0 − A1 /A0 × 100, where A0 is the absorbance of control solution and A1 is the absorbance in the presence of plant extract. The most active extracts (% inhibition ≥ 50%) were assayed for half-inhibitory activity (IC50 ) and assayed for anti-inflammatory activity in vitro and in vivo. IC50 was determined from a graph in which scavenging activity was plotted against varying concentrations (25–125 ␮g/mL) of extract using a linear regression curve.

2.2. Plant material Fresh flower buds of P. nigra were collected in Spring in pollution-free areas and far from towns and plantations in the forest of Tizi Neftah, Province of Amizour, Department of Bejaia (Algeria). The plant was identified in the Laboratory of Botany, University of Bejaia (Algeria). 2.3. Bacterial and fungal strains Gram-negative strains Escherichia coli (O111B4), Pseudomonas aeruginosa (165RIS) and Klebsiella pneumoniaea (TE47), Grampositive strains Staphylococcus aureus (209P) and Bacilus subtilis (ATCC635335) and pathogenic fungi Aspergilus niger (939N) and Fusarium polyferatum were tested. Except for P. aeruginosa (165RIS) and S. aureus (209P) which were donated by Pasteur Institute (Algiers, Algeria), all strains were generously provided by Professor Aziz TOUATI (University of Bejaia). 2.4. Laboratory animals Albino mice of either sex weighing around 23 g were purchased from Pasteur Institute (Algiers, Algeria). Animals were provided with standard food and water ad libitum and were maintained at a constant temperature of 23 ± 1 ◦ C, relative humidity of 65 ± 5% and 12/12 h light/dark cycle. They were weighed, randomized into groups (n = 8), and kept for 2 weeks to acclimatize to laboratory conditions. Experiments were conducted in strict compliance with internationally accepted principles for laboratory animals (Directive of the European Council 86/609/EC).

2.7.1. Diphenylpicrylhydrazil (DPPH) radical scavenging activity Radical scavenging activity of P. nigra flower buds extracts against the stable synthetic radical, diphenylpicrylhydrazil (DPPH), was determined spectrophotometrically using the method of Masuda et al. (1999). Fifty microliters of a solution of DPPH (5 mM) dissolved in methanol were added to 4.9 mL of test sample (100 ␮g/mL) or reference compound. After 30 min of incubation at room temperature, the absorbance was recorded at 517 nm. 2.7.2. ABTS scavenging activity Antioxidant capacity was measured based on the scavenging of ABTS•+ radical cation (Re et al., 1999). Solutions of ABTS (7 mM) and potassium persulfate (2.45 mM) were mixed and incubated in the dark at room temperature for 12–16 h. The product was diluted in ethanol for optimal absorption ±0.7 at 734 nm. The reduction between ABTS.+ and test sample (100 ␮g/mL) was monitored by a decrease in absorption at 734 nm during 30 min. Caffeic acid, quercetin and BHA were used as standards.

2.5. Plant material and extraction procedure

2.7.3. Hydrogen peroxide scavenging activity The ability of the extracts to scavenge hydrogen peroxide (H2 O2 ) was determined using the method of Ruch et al. (1989). Briefly, 2 mL of the test sample or reference compound (100 ␮g/mL) dissolved in methanol were added to 2 mL of a H2 O2 solution (40 mM in phosphate buffer, pH 7.4). After an incubation of 10 min, the absorbance was read at 230 nm.

The fresh flower buds were air-dried in the shade and ground to a fine powder (diameter 63 ␮m). Phenolic compounds were extracted using previously described procedure (Atmani et al., 2009). Ground powder was macerated in ethanol (1:3; w/v) to obtain a crude extract which was dried and subjected to a partition in ethyl acetate and water (1:3:1; w/v/v) to yield two separate fractions (the organic fraction and the aqueous fraction of ethyl acetate). Two equal amounts of the organic fraction of ethyl acetate were further fractionated using hexane and water (1:3:1; w/v/v) on one hand and chloroform and water (1:3:1; w/v/v) on the other

2.7.4. Hydroxyl radical scavenging activity Hydroxyl radical (OH− ) scavenging activity was assayed as described by Halliwell and Gutteridge (1985). Hydroxyl radical was generated in the presence of Fe3+ , EDTA, ascorbate and H2 O2 and monitored by evaluating hydroxyl radical-induced deoxyribose degradation. The reaction mixture contained, in a final volume of 2 mL, 2-deoxy-2-ribose (2.8 mM); KH2 PO4 –KOH buffer (20 mM, pH 7.4); FeCl3 (100 ␮M); EDTA (100 ␮M); H2 O2 (1.0 mM); ascorbic acid (100 ␮M) and (100 ␮g/mL) of the test sample. After incubation for 1 h at 37 ◦ C, 0.5 mL of the reaction mixture was added to

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1 mL TCA (2%) followed by 1 mL TBA (1%). A pink color developed after heating to 100 ◦ C for 15 min. After cooling, the absorbance was measured at 532 nm.

2.7.5. Hypochlorous acid scavenging activity This assay, based on the HOCl-mediated oxidation of thionitrobenzoic acid (TNB) to dithionitrobenzoic acid (DTNB) was performed as described by Hazra et al. (2010). The reaction mixture (3 mL) which contained buffer and a solution of TNB (70 ␮M), with or without extract or reference compound (100 ␮g/mL) was incubated at room temperature. The absorbance was measured at 412 nm before and 5 min after hypochlorous acid (40 ␮M) addition. The amount of TNB not oxidized after incubation, is calculated and expressed as percentage of the TNB initial concentration value.

2.7.6. Nitric oxide scavenging activity The reaction mixture (3 mL) containing 2 mL of sodium nitroprusside (10 mM) and 0.5 mL of phosphate buffer (1 M) was mixed with extract solution (100 ␮g/mL) and incubated at 25 ◦ C for 150 min. 0.5 mL of the reaction mixture containing nitrite was mixed with 1 mL of sulphanilic acid reagent (0.33%) and allowed to stand for 5 min for complete diazotization. Then, 1 mL of naphthylethylenediamine dihydrochloride (1% NEDD) was added and the solution was mixed and allowed to stand for 30 min. Sodium nitroprusside in aqueous solution at physiological pH spontaneously generates nitric oxide, which interacts with oxygen to produce nitrite ions which can be estimated by the use of Griess reagent at 540 nm (Marcocci et al., 1994). Standards (catechin, caffeic acid and quercetin) were used as positive control.

2.7.7. Inhibition of lipid peroxidation The antioxidant activity of extract was determined according to the thiocyanate method of Osawa and Namiki (1981). Plant extracts (2.0 mL) were mixed with 2.05 mL of linoleic acid (2.51%, w/v) in ethanol, 4.0 mL of phosphate buffer (0.05 M, pH 7.0) and 1.95 mL of distilled water and incubated at 40 ◦ C. Every 24 h, 50 ␮L of this mixture were withdrawn and supplemented with 4.85 mL ethanol (75%) and 50 ␮L ammonium thiocyanate. The absorbance was recorded at 500 nm for 3 min after addition of 50 ␮L of ferrous chloride (0.02 M), prepared in hydrochloric acid (3.5%). This procedure was repeated until the absorbance of the blank has reached its maximal value (96 h). The percentage inhibition of peroxidation was calculated as follows: % inhibition of peroxidation = 100 − [(increase in absorbance of extract/increase in absorbance of control) × 100]. 2.8. Anti-inflammatory activity 2.8.1. In vitro anti-inflammatory activity: inhibition of xanthine oxidase Xanthine oxidase (XO)-inhibitory activity was assayed by monitoring the decrease in uric acid formation at 295 nm, using enzyme purified from bovine milk (Atmani et al., 2004). The reaction mixture consisted of 1760 ␮L of sodium-bicine buffer (50 mM, pH 8.3), 200 ␮L xanthine (1 mM), 20 ␮L of (100 ␮g/mL) of the test sample solutions or allopurinol dissolved in dimethylsulfoxide (DMSO) and 20 ␮L of XO solution. XO inhibition was calculated using the following formula: % inhibition = [1 − [At /Ac ]] × 100, where At and Ac are the variation in absorbance of the test solution with and without extract, respectively. XO-inhibitory mode of P. nigra extract that exhibited the best inhibition (% inhibition > 50%) was determined by the Lineweaver–Burk plot using varying concentrations of xanthine (25, 50, 75, 100 and 125 ␮M).

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2.8.2. In vivo anti-inflammatory activity: carrageenan-induced mouse paw edema The animals (n = 8) were fasted for 18 h prior to the experiment and deprived of water only during the experiment. The animals were given orally a dose (200 mg/kg) of the extract (dissolved in 0.9% NaCl). One hour later, 0.05 mL of 0.5% carrageenan suspension in 0.9% NaCl solution was injected into the sub-plantar region of the right hind paw (Winter et al., 1962). Negative and positive control groups received vehicle (10 mL/kg) and sodium diclofenac (50 mg/kg), respectively. For the assessment of the anti-inflammatory activity, paw diameter was measured before and hourly for 6 and 24 h after the carrageenan treatment. Increase in the diameter of the right hind paw was taken as an indicator of edema. The average paw swelling in the group of extract treated-mice was compared to the control and standard groups. Percentage inhibition of edema was calculated according to the following formula: % inhibition = 100 − (mean drug treated group/mean control group) × 100. 2.9. Antimicrobial activity Bacterial strains were grown overnight at 37 ◦ C in Mueller–Hinton Broth (Merck). Fungi were cultured in Sabouraud Dextrose Broth (Fluka) and grown for 48 h at 30 ◦ C. Antibacterial activity of P. nigra extracts was determined by the agar disk diffusion method according to Rubio et al. (2003). A suspension of each microorganism (1 mL) (106 CFU/mL) was carefully mixed in a tube with 18 mL of molten agar and then pipetted into the appropriately labeled Petri dishes. Sterile filter-paper discs (Whatman, diameter 1.6 mm) were impregnated with extracts in methanolic solutions (1 mg/mL), placed in the inoculated plates and incubated at 37 ◦ C for 24 h. Chloramphenicol and Gentamycine, dissolved in the same solvent, were used as positive control while methanol was used as negative control. Concerning antifungal activity, P. nigra extracts were screened by agar well diffusion method (Perez et al., 1990). After 48 h of growth on Sabouraud Dextrose Broth (SDB), fungal cultures were used for inoculation. An aliquot (0.02 mL) (106 spores/mL) was introduced to molten SDB and poured into a Petri dish. After solidification, appropriate wells were made in the agar plate using a 5.0 mm sterile cork borer, into which 0.05 mL of methanolic extracts was introduced. After 24–48 h of incubation at 28 ◦ C, antibacterial and antifungal activities were evaluated by measuring inhibition zones surrounding plant extracts using antibiotic zone scale. 2.10. Statistical analysis The in vitro and in vivo experimental data obtained were expressed as the mean ± SD and mean ± SEM, respectively. The results were computed using analysis of variance (ANOVA) by STATISTICA 5.5 software. Differences were considered to be significant at p < 0.05. The IC50 values were calculated using the OriginPro7.5 software. 3. Results 3.1. Quantification of phenolic compounds The content of various extracts in total phenols, flavonoids and tannins are presented in Table 1. The amount of total phenolics ranged from 14.5 to 114.46 mg catechin equiv./g of extract, the highest being that of the aqueous fraction of chloroform extract. On the other hand, it can be noticed that the organic extracts are rich in tannins, especially the chloroform fraction (430.22 ± 27.63 mg tannic acid equiv./g), compared to their aqueous counterparts

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Table 1 Determination of total phenols, flavonoids and tannins in Populus nigra extracts. Extracts

Total phenolics (mg catechin equiv./g of extract)

Flavonoids (mg quercetin equiv./g of extract)

Tannin (mg ac tannic equiv./g of extract)

Ethanol Ethyl acetate Aqueous of ethyl acetate Hexane Aqueous of hexane Chloroform Aqueous of chloroform

51.78 ± 04.56b 44.15 ± 00.48b 24.43 ± 01.42c 14.5 ± 08.8c ND 25.73 ± 19.15c 114.46 ± 07.7a

13.67 ± 00.34d 25.95 ± 02.29b 32.75 ± 00.28a 07.49 ± 00.92e ND 02.00 ± 00.51f 20.46 ± 00.39c

228.72 ± 26.9c 396.81 ± 31.03b 114.15 ± 28.33d 404.02 ± 33.17a ND 430.22 ± 27.63b 154.47 ± 32.23d

Values are mean ± standard deviation. Analysis of variance (ANOVA) revealed significant effect (p < 0.05)

Fig. 2. ABTS scavenging activity of Populus nigra extracts at 100 ␮g/mL. 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: quercetine; 9: caffeic acid; 10: BHA. Fig. 1. DPPH scavenging activity of Populus nigra extracts at 100 ␮g/mL. 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: BHA.

(aqueous of chloroform 154.47 ± 32.23 tannic acid equiv./g of extract). 3.2. Antioxidant activities The antioxidant potential of P. nigra extracts were assayed using several radicals. In fact, specific radical scavenging assays were used to obtain the necessary information that could be related directly to antioxidant activities of plant extracts. 3.2.1. DPPH scavenging activity As shown in Fig. 1, the highest DPPH scavenging activity was displayed by the chloroform-derived aqueous fraction (65.81 ± 3.07%, IC50 = 24.61 ␮g/mL) which is significant, but remains lower than that of BHA (88.98% ± 1.08%, IC50 = 6.18 ␮g/mL). All other extracts showed a scavenging activity lower than 50%.

Fig. 3. H2 O2 scavenging activity of Populus nigra extracts at 100 ␮g/mL.1: ethanol; 2: ethyl acetate; 3: aqueous ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: BHA; 9: ␣-tocopherol.

scavenger (47.51 ± 0.86%) (Fig. 3), surpassing largely (p < 0.05) the standards BHA (32.98 ± 0.35%) and ␣-tocopherol (28.08 ± 0.39%).

3.2.2. ABTS scavenging activity In the case of the ABTS scavenging activity, the aqueous fractions of chloroform and hexane were found to be the most effective with comparable (p > 0.05) potencies (72.32 ± 1.86%, IC50 = 17.09 ␮g/mL and 73.66 ± 4.26%, IC50 = 14.67 ␮g/mL), respectively (Fig. 2). Nevertheless, standards such as caffeic acid (IC50 = 1.25 ␮g/mL), quercetin (IC50 = 2.26 ␮g/mL) and BHA (IC50 = 3.90 ␮g/mL) have reduced the ABTS radical cation more efficiently in a concentration-dependent manner.

3.2.4. OH. scavenging activity Once again, the aqueous fraction of chloroform expressed the best scavenging activity (47.63 ± 3.59%, IC50 = 113.79 ␮g/mL) against OH. , a deleterious radical. Indeed, this activity was much higher (p < 0.05) than that of catechin (37.85 ± 2.21%, IC50 = 141.78 ␮g/mL) and mannitol (19.58 ± 1.88%, 281.15 ␮g/mL), used as standards (Fig. 4).

3.2.3. H2 O2 scavenging activity Unlike the ABTS and DPPH activities, most extracts demonstrated a weak H2 O2 scavenging activity (lower than 30%). In fact, the aqueous of chloroform fraction was the only potent H2 O2

3.2.5. HOCl. scavenger activity HOCl. -induced oxidation of TNB was efficiently prevented by the aqueous fractions of chloroform and hexane at 100 ␮g/mL (51.42 ± 5.86%, IC50 = 187.90 ␮g/mL) and (50.28 ± 6.13%),

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Fig. 4. Hydroxyl radical scavenging activity of Populus nigra extracts at 100 ␮g/mL. 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: catechin; 9: mannitol.

Fig. 5. HOCl scavenging activity of Populus nigra extracts at 100 ␮g/mL. 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: tannic acid; 9: cathechine; 10: rutine.

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Fig. 6. NO scavenging activity of Populus nigra extracts at 100 ␮g/mL. 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: caffeic acid; 9: quercetin; 10: BHA.

Fig. 7. Inhibition of lipid peroxidation by Populus nigra extracts at 100 ␮g/mL.1: ethanol; 2: ethyl acetate; 3: aqueous ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: BHA.

respectively (Fig. 5), comparable to that of catechin (60.02 ± 1.37%, IC50 = 63.26 ␮g/mL). 3.2.6. NO scavenging activity It is well documented that NO radical is produced in pathogenic conditions such as inflammation. The scavenging activity against NO exhibited by chloroform and hexane aqueous fractions (76 ± 0.20%, IC50 = 9.52 ␮g/mL and 72.86 ± 1.75%) respectively (Fig. 6) was outstanding. Moreover, this activity was found to be similar (p > 0.05) to that of caffeic acid (75.21 ± 3.03%, IC50 = 9.07 ␮g/mL) and significantly (p < 0.05) superior to that of quercetin (66.18 ± 0.04%, IC50 = 12.50 ␮g/mL) and catechin (64.66 ± 1.00%, IC50 = 16.66 ␮g/mL). 3.2.7. Inhibition of lipid peroxidation Except for the aqueous phase of ethyl acetate, all the other P. nigra extracts have demonstrated an appreciable inhibition of linoleic acid peroxidation (Fig. 7) with percentages ranging from 83.4 ± 0.38–93.50.4%.

Fig. 8. XO inhibition by Populus nigra extracts at 100 ␮g/mL. 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: allopurinol.

3.3. Anti-inflammatory activity 3.3.1. Inhibition of xanthine oxidase activity The results illustrated in Fig. 8 point out to the fact that P. nigra aqueous phase of chloroform suppressed efficiently XO activity in a dose-dependent manner (64.90 ± 0.84% at 100 ␮g/mL, IC50 = 60.7 ␮g/mL). However, XO-inhibitory activity exhibited by the above extracts is significantly lower (p < 0.05) than that of

allopurinol (98.18% at 100 ␮g/mL; IC50 = 1.07 ␮g/mL), a known molecule used in therapy against gout. In an attempt to gain more insight into the mechanism of action of this extract, the inhibition mode has been investigated. The Lineweaver–Burk plots of the aqueous fraction of chloroform showed a mixed-type inhibition (Fig. 9) in which the constituents

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Fig. 9. Lineweaver–Burk plot inhibition of XO by aqueous phase of chloroform extract of P. nira.

of the extract react with both the enzyme and the attachment site of the substrate, giving a non productive complex. 3.3.2. Inhibition of carragenan-induced paw edema The results of the anti-inflammatory activity (Table 2) indicate that P. nigra aqueous fraction of chloroform (200 mg/kg) significantly (p < 0.05) inhibited edema formation. The anti-inflammatory activity of the tested extract was observed starting from the first hour after carrageenan injection, extending to the 5th hour, suggesting that its mechanism of action may involve multiple anti-inflammatory mediators. Peak inhibitory effect (62.36%) was reached after 2 h of carrageenan administration, which was found to be comparable to that of the anti-inflammatory drug, sodium diclofenac (50 mg/kg; 54.65%) as shown in Table 2. 3.4. Antimicrobial activity The results summarized in Table 3 indicate that the organic extracts exhibited effective antibacterial activity (inhibition zones ranging between 10 and 17 mm) against tested microorganisms, in comparison with Chloramphenicol and Gentamycin (19.82 and 20 mm, respectively). However, all other extracts showed only moderate activity against fungal strains (inhibition zones between 6 and 9 mm). On the other hand, neither strain of microorganism, whether bacterial or fungal, was sensitive to methanol, the solvent used as vehicle. Interestingly, it is the ethanolic extract (1 mg/mL) which showed antibacterial activity with the largest zones of growth inhibition (ranging between 12 and 17 mm) on gram negative B. subtilis (17.40 ± 2.08 mm) as well as high antifungal activity (inhibition zones ranging between 06 and 09 mm). However, K. pneumoniae strain showed complete resistance to this extract. 4. Discussion Phenolic antioxidants are endowed with the capacity of donating hydrogen atoms or accepting electrons, which requires the presence in their structure of one or more OH groups. The extraction method used in this work aimed at concentrating total phenolics and flavonoids, which are hydrosoluble compounds. These phenolic compounds play an important role in radical scavenging (Rice-Evans, 1995), anti-inflammatory (Dudonné et al., 2011), antibacterial (Bankova et al., 1995) and antifungal (Owolabi et al., 2007) activities. In this paper, we studied the antioxidant potential of several extracts of P. nigra flower buds using various standard chemical and biochemical complementary in vitro methods.

The total antioxidant activity estimated from the evaluation of DPPH and ABTS radical scavenging activities depends on the number of hydroxyl groups of phenolic acids (Okawa et al., 2001). The latter compounds are found in aqueous extracts, in total support of our data and previous findings (Atmani et al., 2009; Parmar and Kar, 2009). The ability of aqueous fraction of chloroform extract to scavenge H2 O2 may be attributed to the structural features of its active components which determine their electron donating abilities to H2 O2 , thus neutralizing it to water. In a comprehensive report, Sroka et al. (2005) revealed that, among examined flavonoids, the strongest H2 O2 scavenging activity was shown for eriocitrin, a compound with two hydroxyl groups bound to the B ring in ortho position. This study has reinforced the findings that the specific structure of the extract constituents may be more important than polarity (Atmani et al., 2009). The high scavenging activity of H2 O2 is regarded as crucial since it generates the highly reactive hydroxyl radical by the Fenton reaction. Hydroxyl is involved in lipid peroxidation which affects membrane fluidity, enzymes and receptor activity, leading to apoptosis (Halliwell and Gutteridge, 1985). Moreover, if hydroxyl radical is generated near nucleic acids, it may react with purine and pyrimidine bases and 2-deoxyribose, leading to mutations and carcinogenesis (Halliwell et al., 1988). P. nigra aqueous fraction was more efficient (p < 0.05) than the standards (catechin, and mannitol), tested in the same conditions. However, it is unclear whether this extract has inhibitory activity for OH. formation by acting as iron chelator, or as OH. scavenger. Previous studies have indicated that ethanol extract from Populus tremula abolished the OH. produced in a Fenton-type reaction system and reduced ROS levels (Oka et al., 2007). The production of HOCl by the neutrophil enzyme, myeloperoxidase (MPO), is an integral part of the nonspecific host defense mechanism triggered by opsonized bacteria, but can also destroy healthy tissues (Halliwell et al., 1988). Our results corroborate several investigations on structure–activity relationships that have established that HOCl scavenging activity depends on both the configuration and total number of hydroxyl groups of polar compounds (Santos et al., 2010). Modulation of NO synthesis or action represents a new approach to the treatment of inflammatory and autoimmune diseases (Marcocci et al., 1994). Similarly, based on the findings of Hazra et al. (2010), the aqueous fraction of P. nigra buds extract used in this study can be considered as a potential antioxidant. Obtained results also showed that the inhibition of lipid peroxidation corresponds closely to the high quenching activity of OH by chloroform and hexane aqueous fractions. However, since organic extracts have also shown a strong inhibition of lipid peroxidation, other mechanisms such as iron chelation, may be involved. A report by Warnant et al. (2004) demonstrated a high prevention of lipid peroxidation by many species of Populus, the most prevalent being that of P. nigra, consolidating therefore our findings. Furthermore, Cu++ -induced oxidation of low density lipoprotein (LDL) is strongly suppressed by different aqueous extracts of a drug named “Phytodolor” composed of Fraxinus-Dolidago-Populus (Rohnert et al., 1998). More recently, a mixture of the ester isomers of P-coumarate and 1-O-rutinose which showed inhibition of lipid peroxidation was identified from the acetone extract of P. tremula knotwood (Neacsu et al., 2007). The inhibition of free radical-producing enzymes such as XO is considered as a good in vitro biochemical model to investigate anti-inflammatory activity. P. nigra aqueous fraction of chloroform extract exhibited promising inhibition potential on XO, compared to many plant extracts tested in the same conditions (Berboucha et al., 2010). Therefore, this extract might be a suitable substitute for allopurinol that causes severe adverse effects when used in

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Table 2 Effect of aqueous fraction of Populus nigra buds extract and diclofenac on carrageenan-induced paw edema in mice. Groups

Paw thickness at different time intervals (in mm) % edema inhibition 0H

1H

2H

3H

4H

5H

24H

Control 10 ml/kg P. nigra 200 mg/kg

2.33 ± 0.02 2.36 ± 0.03 2.34 ± 0.01

3.53 ± 0.08 2.79 ± 0.08 62.36%a 2.90 ± 0.03 54.65% a

3.84 ± 0.15 3.15 ± 0.12 45.45%ab 2.97 ± 0.07 55.55%a

3.88 ± 0.08 3.52 ± 0.10 29.29%b 3.07 ± 0.09 49.35%a

3.86 ± 0.07 3.51 ± 0.12 27.11%b 3.13 ± 0.11 43.74%a

3.58 ± 0.12 3.19 ± 0.12

Diclofenac 50 mg/kg

3.26 ± 0.06 2.82 ± 0.06 50.31%a 2.96 ± 0.03 28.72%b

3.10 ± 0.07

Values are mean SEM. p < 0.05; vs. control, Student’s t-test (n = 8). p < 0.05 significantly different from control.

gout treatment (Pacher et al., 2006). The present findings are in agreement with a previous investigation which has demonstrated the strongest inhibitory potential on XO of many Populus species (Havlik et al., 2010). Other extracts from P. tremula (Meyer et al., 1995) or isolated flavonoids from Populus davidiana stem (Zhang et al., 2006) also inhibited considerably xanthine oxidase. The mixed-type inhibition exhibited by aqueous fraction of chloroform was observed in the case of many phenol compounds like caffeic acid (Masayoshi et al., 1985) and quercetin (Berboucha et al., 2010), which were identified in P. nigra extracts (Jerkovic and Mastelic, 2003; Vardar-Ünlü et al., 2008). The essence of biological activity resides in the bioavailability of the phytochemicals and their extensive metabolism in the organism, which highlights the importance of in vivo testing. Carrageenan-induced acute inflammation in animals is the most suitable test procedure to screen anti-inflammatory agents (Winter et al., 1962; Karawya et al., 2010). The results obtained confirm a high anti-inflammatory potential, superior to that found in a recent investigation carried out on the leaves of P. nigra found in Egypt (Karawya et al., 2010). Such efficiency would validate the medicinal use of P. nigra extract against acute inflammatory conditions. Moreover, the presence of copious amounts of quercetin, a known anti-inflammatory agent (Guardia et al., 2001), has already been revealed in P. nigra bud extracts (Vardar-Ünlü et al., 2008). Other compounds found in Populus species can be responsible for their anti-inflammatory properties. Earlier, Shen (1988) reported that populin, isolated from the leaves of P. tomentosa, showed antipyretic and analgesic activities. More recently, Babst et al. (2010) proved that anti-inflammatory and anti-rheumatic properties of Populus species are mainly due to their content in salicylate. The mode of action of the fraction tested is not clear at this point, but it can be suggested that it may act via inhibition of multiple anti-inflammatory mediators, probably through regulation of genes involved in antioxidant defenses and inflammatory response in vitro (Dudonné et al., 2011). The results of antibacterial activity showed that bacteria were more sensitive to organic than water extracts, in accordance with

previous studies (Abu-shanab et al., 2004; Owolabi et al., 2007; Valarmathy et al., 2010). In fact, it has been postulated that all of the identified plant components known for their anti-microbial activity are most often obtained through initial ethanol or methanol extraction (Abu-shanab et al., 2004; Owolabi et al., 2007). Propolis has been reported to be active against Gram-positive bacteria (Grange and Davey, 1990) or potentiates some antibiotic effects (Krol et al., 1993). Gram-positive bacteria S. aureus (209P) and B. subtilis (ATCC635335) were the most susceptible to growth inhibition by the tested plant extracts, in agreement with previous results (Vardar-Ünlü et al., 2008). The greater susceptibility of Gram-positive bacteria has also been previously reported (Nostro et al., 2000). Susceptibility differences between Gram-positive and Gram-negative bacteria may be due to cell wall structural differences between these two classes. Furthermore, it has been shown that anti-bacterial activity could be due to a synergistic action between some phenolic components (Vardar-Ünlü et al., 2008). Alternatively, the lipophilic nature of the buds exudates of P. nigra, which promotes extract penetration into the bacterial membrane, has also been reported (Bankova, 2005). The above findings suggest that the antioxidant activities of extracts tend to be method-specific, as was stated earlier (Parmar and Kar, 2009). This could be due to differences in the mechanisms of action and the specific radical scavenging activity of the extracts studied (Hazra et al., 2010). The antioxidant activity of the tested extracts might have been mediated by the presence of phenolic components. This assumption is further supported by the high content of total phenolic compounds and flavonoids present in the aqueous fraction of chloroform extraction (Table 1). It is also plausible to include the antioxidant property of the plant extract as one of the mechanisms that contributed to the observed anti-inflammatory activity. Tannins, which are found in high amounts in organic extracts (Table 1), might be responsible for antibacterial activity. This assumption is supported by an investigation that implicated catechins and more specifically tannic acid in the destruction of cell membranes of bacteria such as S. aureus (Akiyama et al., 2001).

Table 3 Antimicrobial activity of Populus nigra buds extract against pathogenic microorganisms. Extract 1 mg/mL

Inhibition zone of microorganisms (mm)* E. coli

S. aureus

P. aeruginosa

B. subtilis

K. pneumoniaea

A. niger

F. polyferatum

1 2 3 4 5 6 7 8 9 Methanol

14.26 ± 0.61 12.56 ±0.30 Na Nt Nt 12.96 ± 0.58 Na 20.7 ± 1.54 Nt Na

14.30 ± 0.32 14.5 ± 0.55 Na Nt Nt 14 ± 1.01 Na 24.40 ± 2.53 Nt Na

12.20 ± 0. 60 11.83 ± 1. Na Nt Nt Na Na 19.82 ± 1.33 Nt Na

17.40 ± 2.08 15.30 ± 0.70 Na Nt Nt 12.5 ± 0.95 Na Nt 30.20 ± 1.30 Na

Na 11.90 ± 1.65 08.95 ± 0.21 Nt Nt 10 ± 0.28 12.50 ± 0.70 Nt Na Na

07.85 ± 0.07 Na 06.10 ± 0.14 Nt Nt 07 ± 0.56 06 ± 5.24 Nt Nt Na

06.90 ± 1.21 07.73 ± 1.31 7.53 ± 1.38 Nt Nt 08.06 ± 1.63 06.98 ± 2.21 Nt Nt Na

Nt: not tested; Na: no activity; 1: ethanol; 2: ethyl acetate; 3: aqueous of ethyl acetate; 4: hexane; 5: aqueous of hexane; 6: chloroform; 7: aqueous of chloroform; 8: chloromfenicol; 9: gentamycin. * The average of three values.

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