Mol2Net-04 Assessment of antioxidant and antibacterial properties of - - PDF document

Mol2Net-04, 2018, BIOCHEMPHYS-01 (pages 1- x, type of paper, doi: xxx-xxxx http://sciforum.net/conference/mol2net-4

Mol2Net-04 Assessment of antioxidant and antibacterial properties of some Tunisian plants

Manel Ben Ali1, 2*, Houda Hmani1, Lobna Daoud1, Mouna Jlidi1, Adel Hadj Brahim1 , Samir Bejar1 and Mamdouh Ben Ali1,2

1Laboratory of Microbial Biotechnology and Engineering Enzymes (LBMIE), Center of Biotechnology

• f Sfax (CBS), University of Sfax, Road of Sidi Mansour km 6, PO Box 1177 Sfax 3018, Tunisia

E-Mails: houda_enis@yahoo.fr (Houda hmani); lobna.daoudm@gmail.com (Lobna daoud); jlidimanno@yahoo.fr (Mouna Jlidi); adelhadjibrahim@gmail.com (Adel Hadj brahim); samir.bejar@cbs.rnrt.tn (Samir Bejar)

2Astrum Biotech, Business incubator, Center of Biotechnology of Sfax (CBS), University of Sfax,

Road of Sidi Mansour km 6, PO Box 1177 Sfax 3018, Tunisia; E-Mail: mamdouh.benali@cbs.rnrt.tn; * Corresponding author: E-Mail: manel.benali@gmail.com Received: / Accepted: / Published: Abstract: This study is a screening of antioxidant and antibacterial activities of a multitude of Tunisian plants extracts. Total phenolic and total flavonoid contents were also determined. Results showed that total phenolic and flavonoid contents of the samples as well as antioxidant activities varied greatly among different plant parts and suggest that many plants are rich in antioxidant

• compounds. Interest will be taken to promising active plants particularly those which combine

antioxidant properties with antimicrobial activities. Keywords: Tunisian plants; total phenolics; flavonoids; antioxidant activity; antibacterial activity

• 1. Introduction

Scientists have focused on plants as sources of natural antioxidants since they can protect the human body from free radicals. The antioxidant properties of plant extracts have been attributed to their metabolites Particularly polyphenol contents [1, 2]. Medicinal plant parts (roots, leaves, branches/stems, barks, flowers, and fruits) are commonly rich in phenolic compounds. In addition with the capacity of these compounds to suppress lipid oxidation, the major factor causing food deterioration during storage and processing[3], many studies have reported their antimicrobial activities and some of them are classified as Generally Recognized Safe Substances (GRAS) [4]. The objective of this work was to screen a number of Tunisian plant extracts by evaluating their total phenolics and flavonoid contents, antioxidant (free radical-scavenging and ß- carotene) and antibacterial activities.

• 2. Results and Discussion

2-1. Antioxidant activity by DPPH free radical- scavenging activity Methanol extracts of the studied plants were the most effective DPPH radical scavengers. Twenty eight of the investigated methanol extracts, inhibited DPPH absorption with percentages over 80%. Methanolic extract of Amaranthus hybridus stalks (IP = 19.13%), Citrillus colocynthis fruit (IP = 26.76%) and Chenopodium humile leaves (IP = 33.53%) contained remarkably lower amounts of radical scavenging compounds according to their lower IP values. Ten ethyl

SciForum

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 2 acetate extracts, which are considerably less effective radical scavengers compared to the corresponding methanol extracts, exhibited inhibition percentages higher than 80%. In addition, radical scavenging activities of ethyl acetate extracts of Pelargonium roseum flowers and Salvia officinalis were similar to those of methanol extracts of the same plant parts. However, the ethyl acetate extract of C. humile (IP = 62.03%) was more effective than its methanol extract (IP = 26.76%). Ten ethyl acetate extracts and sixteen methanol extracts have radical scavenging activity with IC50 < 100 µg/ml. free radical-scavenging activity of methanol S. officinalis leaves extract is the strongest ( IC50 = 3.34 µg/ml) tested extract followed by methanol extracts of

• P. roseum leaves (8.25 µg/ml) and stalks (10.4

µg/ml). IC50 value of methanol extract of S.officinalis stalks was also noteworthy (13.04 µg/ml) when compared to the synthetic antioxidant BHT (7.7 µg/ml) used in this study as positive control. 2-2. Determination of antioxidant activity by-β- carotene bleaching method In the β-carotene / linoleic acid system oxidation

• f linoleic acid was effectively inhibited by
• verall tested extracts with inhibition percentages

values greater than 70%. Consequently, there was no much difference in the antioxidant potential of the extracts prepared by increasing of solvent polarity. Both extracts, ethyl acetate of Juncus maritimus (92.86%) and methanol of P. roseum stalks (92.66%), displayed the greatest antioxidative properties in emulsion, while A.hybridus seeds demonstrated the lowest activity (9.029%) at plant material. Different bleaching rates were demonstrated for the common antioxidants, BHT (84.06%) and α-tocopherol (87.27%). When compared to both ethyl acetate and methanol extracts, twelve extracts are stronger in linoleic peroxidation activity than these antioxidants. This result indicates that compounds with the strongest antioxidant activity in the β-carotene-linoleate assay system can be of medium or high polarity. 2-3. Total phenolic and flavonoid contents The amount of Total phenolic Compounds (TPC)

• f the selected plants, ranged from 12.03 to

852.24 mg GAE/g extract, could be categorized into 4 classes: very high (>300 mg GAE/g), medium high (100–300 mg GAE/g), medium low (50-100 mg GAE/g ), low (10-50 mg GAE/ g) and very low ( <10 mg GAE/ g). Most of the extracts showed medium high level

• f TPC. The highest amounts were found in the

methanolic extracts, especially of S. officinalis leaves and flowers (respectively 852.2 mg GAE/g and 557.5 mg GAE/g), P. roseum leaves (891.8 mg GAE/g), R. officinalis leaves (553.5 mg GAE/g) and Artimisia absinthium stalks (588.51 mg GAE/g). It was noticed that some leaves had higher TPC than flowers and stalks of the same species, i.e. TPC

• f

leaves methanolic extracts

• f
• A. hybridus, Atriplex mollis, C. colocynthis,

Marribium alysson, Rosmarinus officinalis, S.

• fficinalis and P. roseum were significantly

higher than their respective flowers and stalks methanolic extracts. Although TPC of Ocimum basilicum flowers extract was found to be higher than that of his corresponding leaves. This

• bservation is valid for stalks ethyl acetate

extracts

• f

R.

• fficinalis,

S.

• fficinalis

and P. roseum. Differences in antioxidant distribution in the leaves, flowers and Stalks could be explained by the following

• bservations. Siddhuraju and coworkers [5] have

reported that the production of anthraquinones, xanthones, flavonol and proanthocyanidins in leaves would lead to higher antioxidant

• activity. Del Bano et al. [6] have found that some

antioxidative compounds were selectively biosynthesized by the leaves but the compounds do not exist in flowers. In addition, it has been reported that the amount of total phenolics vary with respect to families and varieties [7,8]. In this work we calculated the total flavonoid contents in units of mg quercetin equivalent. Existence of higher total flavonoid amount was observed in the methanolic extracts prepared the aerial parts

• f each species. However, in some cases, the

ethyl acetate extract of some plant parts exceeded its methanolic extract. The content of flavonoids (mg/g extract) in quercetin equivalents varied from 1.5 to 184.9 mg Q/g extract. The highest amounts of flavonoids were found in methanolic extracts of A. mollis leaves (166.3), A. mollis stalks (184.9) and A. Tenuifolius flowers (156.9). Lowest amounts are in ethyl acetate extracts of Salsola suaeda (1.8) and C. colocynthis (2.1 mg

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 3 Q/g) leaves. However, extracts with higher phenolic content did not always have a higher flavonoid content, as was evident for example for ethyl acetate extracts

• f

C. colocynthis, methanolic extracts of C. colocynthis fruit and

Hyoscyamus albus flowers and stalks.

According to literature, there is a disagreement about relation between antioxidant activity and total phenolics or flavonoids contents. Many studies approved a good correlation [7,9]. While some authors have observed high correlation,

• thers find no direct correlation or only a very

weak one [10, 11]. In this study, we found no association between total phenolics or flavonoids contents and the activity of the extracts as DPPH scavengers (data not shown) which can due to the diversity of plant material (different plant families and plant parts). However, the number

• f the plant samples tested in previous studies is
• ften very limited. Herein, we systematically

investigated seventy samples representing thirteen different species of Tunisian plants. 2-4. Antimicrobial activity Quantitative evaluation of the antimicrobial activity of ethyl acetate and methanolic extracts

• f the studied Tunisian plants was carried out

against selected microorganisms. Of the 70 analysed extracts except Pituranthos tortuosus ,

Asphodelus tenuifolius, S. suaeda , Juncus

maritimus, Stippa tenacissima , H. albus stalks, thymelea hirsuta and Ruta chalapensis extracts which are inactive toward all strains, twenty seven of the two types of plant extracts showed varied antimicrobial efficacies against all the reference microorganisms. In previous screenings of medicinal plants for antimicrobial activity, most of the active plant extracts showed activity against Gram-positive strains only [12, 13]. These findings correlate with

• ur results. In fact, antimicrobial action of the

Tunisian plant extracts is more pronounced on Gram-positive bacteria particularly S. aureus. However, some extracts have notable action on Gram negative bacteria (P. aeruginosa). Only ethyl acetate extract of S. officinalis stalks and methanolic extract of P. roseum flowers and stalks were active against E. coli and S. enterica respectively. Low MIC values indicate potentially high efficacy of the extracts as antimicrobial agents

[14, 15].

A classification of activity of plant extracts on the basis of their MIC values was proposed by Aligiannis et al. [16]: strong inhibition: MIC < 0.5 mg/mL; moderate inhibition: 0.6 mg/mL < MIC <1.5 mg/mL and low inhibition: MIC > 1.6 mg/mL. Accordingly, most of the studied plant extracts exert a strong inhibitory activity on M. luteus (MIC = 0.078; 0.15; 0.31 mg/mL), on S. aureus (MIC = 0.078; 0.15; 0.31 mg/mL) and a few are

• P. aeruginosa (MIC = 0.15; 0.31 mg/mL). The

rest of active plants have moderate inhibition on different strains (MIC = 0.625 mg/ml) and a few are with low inhibition (MIC = 2.5 mg/ml). As mentioned above the MICs of tested extracts were generally low and all extracts are bactericidal in their tested concentrations and since the plant extracts were in crude form, these results are promising. Among investigated plants, nine extracts contained substances which delay the only yeast assayed C. albicans growth in different concentrations (1.25; 0.625; 0.31; 0.15 mg/ml). Only ethyl acetate extract of S. officinals inhibits C .albicans at very low concentration (0.078 mg/ml). The most effective extract towards the studied microorganisms seems to possess the best antioxidant activity. This finding is interesting and allows us to conclude that the concordance

• f these two biological properties is the result of

the richness extracts in bioactive compounds.

• 3. Materials and Methods

Chemicals were purchased from Sigma Chemical. Eighteen plants used in Tunisia, belonging to thirteen plant families, were selected for this

• study. Plant parts, sorted on flowers, stalks, fruits
• r leaves, were dried at room temperature for 2–3
• weeks. Dried material was chopped and

extracted with ethyl acetate then methanol with

• stirring. After filtration, the combined ethyl

acetate and methanol extracts were evaporated under vacuum to absolute dryness then kept at 4°C for further analysis. Radical scavenging activity of plant extracts against stable DPPH was determined

Mol2Net, 2015, 1(Section A, B, C, etc.), 1- x, type of paper, doi: xxx-xxxx 4 spectrophotometrically. The free radical scavenging activity of extracts by DPPH were examined by comparing to that of known antioxidant BHT using modified method of Miliauskas et al. [17] .The percent of DPPH discoloration of the samples was calculated by the following formula : IP (%) = [(Ablank/Asample)/Ablank] × 100 where Ablank is the absorbance of the control reaction (containing all reagents except the test sample) and Asample is the absorbance of the extracts or the reference. In the Determination of antioxidant activity by-β- carotene bleaching method antioxidant capacity is determined by measuring the inhibition of the volatile oxidation organic compounds and the conjugated diene hydroperoxides arising from linoleic acid [18] .Antioxidant capacities of the samples were compared with those of BHT and the blank. Total soluble phenolic compounds in the extracts were measured according to the method of Singleton and Rossi [19] and expressed as gallic acid equivalents. The total flavonoid content was determined using the Dowd method as adapted by Arvouet-Grand et al.[20] .The total flavonoid content was determined using a standard curve with quercetin Antimicrobial activities of the crude plant extracts were tested against eight strains of bacteria i.e. three Gram-negative strains; Escherichia coli (ATCC 8739), Pseudomonas aeruginosa (ATCC 49189) and Salmonella enterica (ATCC 43972), and four Gram-positive strains; Micrococcus luteus (LB 14110), Staphylococcus aureus (ATCC 6538), Enterococcus faecalis and Listeria monocytogenes obtained with one yeast Candida albicans from the Microorganisms Collection of the Laboratory

• f

Microorganisms and Biomolecules in the Center of Biotechnology of Sfax-Tunisia. All experiments were conducted in triplicate and results are expressed as mean ± standard deviation (SD).

• 4. Conclusions

The results obtained in this study, showed a noteworthy difference in antioxidant activity, antimicrobial propriety and polyphenol amounts among the studied Tunisian plant parts. Furthermore, the study of antimicrobial activity of all extracts toward different strains showed that many plant extracts can act as radical scavengers, peroxidation protectors or antibiotics as a certain extent. Further work is to confirm these effects of the promising plants by using other assays with different mechanism actions particularly for antioxidant capacity. Our objective will be to isolate and identify natural compounds responsible of such activities and discussion the possibility of their reintroduction in foods system or their use in the the prevention and therapies of diseases in which oxidants or free radicals are involved. Acknowledgments This research was supported by the ministry of high education and scientific research. References and Notes 1. Heim, K. E., Tagliaferro, A. R., Bobilya, D. J. Flavonoid antioxidants: Chemistry, metabolism and structure-activityrelationships. The Journal of Nutritional Biochemistry. 2002, 13, 572–584. 2. Allouche, N., Hamden, K., Damak. M., Elfeki, A. Hypoglycemic and antioxidant effects of phenolic extracts and purified hydroxytyrosol from olive mill waste in vitro and in rats. Chemico-Biological Interactions. 2009, 180, 421-432. 3. Frankel, E.N., Huang, S.W., Aeschbach, R. Antioxidant activity of green teas in different lipid

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