Scholarly article on topic 'Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties'

Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties Academic research paper on "Chemical sciences"

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Abstract of research paper on Chemical sciences, author of scientific article — Eimad dine Tariq Bouhlali, Mohamed Bammou, Khalid Sellam, Mohamed Benlyas, Chakib Alem, et al.

Abstract The research aimed to examine the antioxidant, anti-hemolytic and antimicrobial activities of six Moroccan date fruit varieties. Estimation of total phenolic and flavonoid contents revealed that, Bousrdoun (537.07mg GAE/100g DW) and Jihl (208.53mgRE/100g) had the highest phenolic and flavonoid contents, respectively. Among the date fruit varieties tested for antioxidant activities, Jihl had the highest activity compared to other varieties. It had an inhibitory concentration (IC50) value of 2.05g/L for DPPH scavenging activity and a ferric reducing power of (860.89μmolTE/100gDW). As well as a high protective effect against AAPH-induced erythrocyte hemolysis with a hemolysis half-time of 210.99min. The antibacterial capacity of various extracts was investigated against Gram positive (Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 29213, Staphylococcus aureus ATCC 25923) and Gram negative bacteria (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella abony NCTC 6017). The Bousrdon and Jihl extracts were found to be more potent inhibitory activities with MIC values ranging between 2.5mgmL− 1 and 10mgmL−1 for all bacterial strains tested. These results suggested that date fruit extract, especially Jihl and Bousrdon extract, is not only an important source of antioxidants, which possess a high protective effect of membrane against free radical, but also a potential source of antibacterial components.

Academic research paper on topic "Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties"

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Original article

Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties

Eimad dine Tariq Bouhlali, Mohamed Bammou, Khalid Sellam, Mohamed Benlyas, Chakib Alem, Younes Filali-Zegzouti

PII: DOI:

Reference:

S1018-3647(15)30015-X http://dx.doi.org/10.1016/j.jksus.2016.01.002 JKSUS 360

To appear in:

Journal of King Saud University - Science

Received Date: Accepted Date:

29 August 2015 10 January 2016

Journal of

King Saud University

-Science King Saud University

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Please cite this article as: E.d.T. Bouhlali, M. Bammou, K. Sellam, M. Benlyas, C. Alem, Y. Filali-Zegzouti, Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties, Journal of King Saud University - Science (2016), doi: http://dx.doi.org/10.1016/jjksus.2016.01.002

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Evaluation of antioxidant, antihemolytic and antibacterial potential of six Moroccan date fruit (Phoenix dactylifera L.) varieties.

Eimad dine Tariq Bouhlaliab, Mohamed Bammoua , Khalid Sellama, Mohamed Benlyasa, Chakib Alemb, and Younes Filali-Zegzoutia.

a Team of Biology, Environment and Health, Faculty of Science and Technology Errachidia. Morocco, b Team of Biochemistry of Natural Substances, Faculty of Science and Technology Errachidia. Morocco Corresponding author: Eimad dine Tariq BOUHLALI

Phone: +212698984535.

Address: Biochemical of natural resources laboratory, Faculty of Science and Technology Errachidia 52000. Morocco

Email: bouhlali.eimad@gmail.com

ighest îd for

Abstract

The research aimed to examine the antioxidant, anti-hemolytic and antimicrobial activities of six Moroccan date fruit varieties. Estimation of total phenolic and flavonoid contents revealed that, Bousrdoun (537.07 mg GAE/100 g DW) and Jihl (208.53 mg RE/100 g) had the highest phenolic and flavonoid contents, respectively. Among the date fruit varieties tested antioxidant activities, Jihl had the highest activity compared to other varieties. It had an Inhibitory Concentration (IC50) value of 2.05 g/L for DPPH scavenging activity and a ferric reducing power of (860.89 ^mol TE/100g DW). As well as a high protective effect against AAPH-induced erythrocyte hemolysis with a hemolysis half-time of 210.99 min. The antibacterial capacity of various extracts was investigated against Gram positive (Bacillus subtilis ATCC 6633, Bacillus cereus ATCC 29213, Staphylococcus aureus ATCC 25923) and Gram negative bacteria (Echerichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Salmonella abony NCTC 6017). The Bousrdon and Jihl extracts were found to be more potent inhibitory activities with MIC values ranging between 2.5 mg.mL1 and 10 mg. mL-1 for all bacterial strains tested.

These results suggested that date fruit extract, especially Jihl and Bousrdon extract, is not only an important source of antioxidants, which possess a high protective effect of membrane against free radical, but also a potential source of antibacterial components.

Keywords: date fruit, antioxidant, anti-hemolytic, antibacterial.

1. Introduction

Date fruit is one of the oldest known fruit crops in southeast Morocco, where it is considered as an ideal food because of its edible sweet fruit, energy boosters and hunger pacifiers also, due to of its many medicinal properties. Date fruit contains a wide range of chemical compound such as phenolic acid, flavonoids, anthocyanins, procyanidins, carotenoids, and sterols, which is known to have too many health benefits (Baliga et al., 2011). The pulp and the pit of date fruit have been used in traditional medicine for the prevention of a wide range of infectious diseases. The ethnomedicinal inspection showed that people in southeast Morocco used date fruit to treat hypertension and diabetes (Tahraoui et al., 2007). The date fruit known in the folk medicine for its laxative effect and it is used also to regulate the urine and in vaginal pessaries, to improve sexual potency and fertility, also to cure abdominal complaints, vomiting, asthma, bronchitis, fever, fatigue, tuberculosis, wandering of the mind and loss of consciousness. (Aamir et al., 2013; Ammar et al., 2009)

Pre-clinical researches that were done using date fruit extract shown a wide spectrum of health benefit such as antioxidant, antimutagenic, antimicrobial, anticancer, anti-inflammatory, gastroprotective, hepatoprotective, nephroprotective, and immunostimulant activities (Baliga et al., 2011).

The target of this research is to investigate the antioxidant, anti-hemolytic and antibacterial potential of six Moroccan date varieties.

2. Materials and methods

Six Moroccan date varieties locally known as Bouskri, Bousrdon, Bousthammi, Boufgous, Jihl and Majhoul considered to be the premium quality in the south-east Morocco were obtained at Tamr stage (October 2014) from The Errachidia National Institute for Agricultural Research. The samples were rinsed, pitted and stored at -20 °C until extraction and analysis.

2.1. Preparation of rich polyphenol extracts

The rich Phenolic compound extract was prepared according to the method of Bouhlali et al, (2015c). Briefly, 30 g of pitted and crushed date fruit were extracted with 150 mL methanol-water (4:1, v/v), at 35 °C for 12 h using an orbital shaker-incubator. The mixture was then filtered and the filtrate was concentrated under reduced pressure at 40 °C until the total

evaporation of solvent using a rotary evaporator. The results methanolic crude extract were kept at -20 °C in dark glass bottles until use.

2.2. Measurement of total polyphenolic contents

The total phenolic contents of date fruit extract were measured according to the method of Bouhlali et al, (2015b). Briefly, 500 ^L of Folin-Ciocalteau reagent (10-fold diluted with water) were mixed with 100 ^L of date fruit solution, and then 400 ^L of aqueous sodium carbonate solution (7.5 % w/v) were added. The mixture was allowed for 60 min at room temperature and measured the absorbance at 765 nm. The calibration curve was prepared using Gallic acid. The total phenolic compounds were expressed as Gallic acid equivalent in mg/100 g dry weight (DW) of date fruit.

2.3. Measurement of flavonoid content

leterminei

The total flavonoid content of date fruit extract was determined by the method of Kim et al, (2003). One mL of date fruit solution was mixed with 4 mL of distilled water. Then 300 ^L of aqueous sodium nitrite solution (5%) were added, followed by 300 ^L of aqueous aluminum chloride solution (10 %). Test tubes were incubated for 5 min at room temperature, and then 2 mL of sodium hydroxide (1 M) were added to the mixture and then the final volume was made up to 10 mL with distilled water. The mixture was vortexed and the absorbance was determined at 510 nm. The absorbance was calibrated to a standard curve of Rutin solution and the results were expressed as mg Rutin equivalents (RE)/100 of dry weight (DW) of date fruit.

2.4. Measurement of total condensed tannins

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(2006). solution me to P

The total condensed tannins were determined using the method described by Heimler et al, (2006). Concisely 400 ^L of the date fruit extract were mixed with 3 mL of methanolic of vanillin (4 %) and 1.5 mL of concentrated hydrochloric acid. The absorbance was asured at 500 nm after incubation at room temperature for 15 min. The catechin was used prepare a calibration curve, and the results were expressed as mg CE (catechin equivalents) /100 g of dry weight (DW) date fruit.

2.5. ABTS radical scavenging assay

The ABTS assay was done using the method of Re et al, (1999). The ABTS radical cations (ABTS+) were produced by reacting aqueous solution of ABTS (7 mM) with an aqueous

solution of potassium persulphate (2.45 mM). The mixture was left to at an ambient temperature in the dark for 12-16 hours before use, the result solution was diluted with distilled water to obtain 0.700 ± 0.005 at 734 nm in the absorbance. 30 ^L of the sample added to 3 mL of the ABTS solution were allowed at an ambient temperature. After 6 min the absorbance at 734 nm was recorded immediately. Trolox solution was used to prepare the standard curve and the total antioxidants were expressed as mmol of Trolox equivalents per 100 g of dry weight (DW) of date fruit.

2.6. DPPH radical scavenging activity

Radical scavenging activity of date fruit extract against stable DPPH was evaluated as described by Blois (1958) method with slight modifications. The reaction mixture contained 100 ^L of date fruit extract at a different concentration and 1.9 mL of methanolic DPPH (0.3 mM). The result mixtures were left at room temperature for 20 min and the absorbance was measured at 517 nm. The IC50 (concentration providing 50 % inhibition) values were calculated from the plotted graph of scavenging activity against the concentrations of the samples.

Abs control is the absorbance without extract; Abs sample is the absorbance of the extract or standard.

2.7. Ferric reducing antioxidant power assay

ing activ

wer blan

The ferric reducing activity of date fruit extract was estimated based on the method of Benzie and Strain, (1999). The FRAP reagent was prepared by mixing 50 mL of acetate buffer (0.3 M ) at (pH 3.6), 5 mL tripydyltriazine (TPTZ) solution 10 mM prepared in HCl (40 mM) and 5 mL of Ferric chloride solution (FeCl3) (20 mM). 2 mL of the freshly prepared FRAP reagent e added to the 10 ^L of extract. Then the absorbance was measured at 593 nm against the blank after 10 minutes at ambient temperature. Trolox was used to prepare the standard curve. The result was expressed as Trolox equivalent in mmol/100 g of dry weight (DW) of date fruit.

2.7. Evaluation of protective effect date fruit extract against AAPH induced erythrocyte oxidative hemolysis

The anti-hemolytic activity induced by a peroxyl radical initiator, AAPH was measured according to the method established by Bouhlali et al., (2015a). Two hundred microliters of Rabbit blood collected in heparin bulbs were mixed with 10 ^L of date fruit extract, and then 600 ^L of AAPH (10 %) were added. The mixture was incubated at 37 °C. The absorbance of the mixture was measured at 450 nm every 5 min. The date fruit extract was replaced by Trolox and saline (0.9 % NaCl) in the positive and the negative control respectively. The Protective effects of date fruit extract on free radical induced hemolysis of erythrocytes were estimated from the time required for half-hemolysis.

2.8. Antibacterial activity

2.8.1. Bacterial strains

The antimicrobial activity was evaluated against six selected bacteria associated with various forms of diseases, three Gram-positive: Bacillus cereus ATCC 29213, Staphylococcus aureus ATCC 25923, Bacillus subtilis ATCC 6633 and three Gram negative: Pseudomonas aeruginosa ATCC 27853, Echerichia coli ATCC 25922, Salmonella abony NCTC 6017. Microorganisms were obtained from the culture collection of The National Institute of Hygiene (Rabat).

The disc-diffusion test of date fruits extracts was carried out using the method described by the National Committee for Clinical Laboratory Standards (NCCLS, 2002) with slight modification. Briefly 250 ^L of bacterial cell suspension (concentration of 108 CFU/mL prepared using an overnight culture growth incubated at 37°C) was spread over the plates containing Mueller Hinton agar media, then a paper disc impregnated with 15 ^l of the different date fruit extracts was placed on the surface of the media. Then the diameter of an inhibition zone was measured around the discs after incubation of Petri dishes for 24 h in 37 entamicin (10 ^g/disc) a known antibiotic drug was used to control the sensitivity of the tested bacteria. One other Petri dish was used as a negative control contains the bacterial cell suspension without the test extract. This assay was done in triplicates, and the inhibition zones of the extract were compared with those of reference antibiotic.

2.8.2. Disc-diffusion method

inhibiti °C. Gei tested b

2.8.3. Minimum Inhibitory Concentration (MIC)

The antibacterial activity of the various date fruit extract was tested using the micro-dilution method of Drummond and Waigh (2000) with a slight modification. First of all, the resazurin solution was prepared by dissolving one tablet in 40 mL of sterile water and the bacterial culture was prepared by diluting an overnight culture of a test bacterium in 0.1 % physiologic water to obtain 106 colony forming units/mL. Afterwards 100 ^L of bacterial culture were added in each numbered wells followed by 100 ^L of resazurin solution and then 100 ^L of each serially diluted date fruit extract was added in well. After incubation in 37 oC for 6 hours, the blue colored solution in microtiter plates indicates growth inhibition of bacteria. The negative control contained the sterile broth and resazurin solution, although the positive control is made up of broth culture and resazurin solution.

2.9. Statistical analysis

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Statistical analysis was performed using StatView 5.0 software. The experimental results were reported as mean ± SE (standard error) (n = 6) on a dry weight for all experiment except antibacterial activity where the results were reported as mean ± SD (standard deviation) (n = 3). The analysis of variance (ANOVA) and the post-hoc Bonferroni. (p < 0.0018) tests were used to compare the experimental groups. Pearson's correlation coefficient (r) was used to measure the association between two variables. Differences at p < 0.05 were considered significant.

3. Results and discussio

3.1. Phenolic content

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The total phenolic contents of date fruit varieties were estimated using Folin-Ciocalteu's colorimetric method, which is simple, rapid, reproducible and low-cost method. This method relies on the reduction of phosphomolybdic-phosphotungstic acid in an alkaline medium in the presence of phenolic compounds (Fu et al., 2011). Table 1 showed significant differences within the analyzed date fruit varieties (p < 0.05). The highest value of total polyphenols was determined in Bousrdon (537.07 mg GAE/100 g DW) and the lowest level was found in Bouskri (331.86 mg GAE/100g DW). The polyphenolic content in this study was higher compared to the study of Hasnaoui et al. (2012) for the Moroccan varieties who found the total phenolic content ranged between 171.4 and 353.92 mg GAE/100 g DW. Our results confirm previous results established by Lemine et al, (2014) (405.5 - 661.1 mg GAE/100g DW) for Mauritanian date cultivars and Benmeddour et al, (2013) (226 to 955 mg GAE/100 g

DW) for Algerian date cultivars. These variations in the total phenolic contents may depend on varieties, maturity growing condition, storage conditions, fertilizer, soil type, season, geographic origin and amount of sunlight received may also be critical in this respect (Al-Farsi et al., 2007; Besbes et al., 2009).

3.2. Flavonoids content

Table 1 summarized that the flavonoid content in analysed date fruits varied widely and ranged from 68.88 to 208.53 mg RE /100 g DW. Jihl cultivar showed the highest amount of flavonoid contents and Bouskri cultivar showed the lowest level. Our results are largely in good concordance with those previously announced by Benmeddour et al, (2013) and Lemine et al, (2014) who established that the total flavonoid content on dried matter of date fruit at the same stage of maturation "Tamr" ranged from 15.22 to 299.74 mg QE/100 g and 39.5 -

112.5 mg QE/100 g respectively. However, our observed results are very higher than those reported by Biglari et al, (2008) who found that the total flavonoid content in the Iranian date cultivars varied from 1.62 to 81.79 mg CE/100g DW.

Flavonoids are known to be synthesized by plants in response to microbial infection; hence, it should not be astonishing that they have been found in vitro to be effective antimicrobial substances against a wide array of microorganisms (Kumar and Pandey., 2013). Several scientific investigation showed that flavonoid possess a wide range of health benefits, which include oxidative stress such as anti-allergic, anti-inflammatory, analgesic, and cardio protective, hepatoprotective, anticancer, antiviral activities. This variability of therapeutic potentials depend on flavonoid structure (Agrawal, 2011; Kumar and Pandey, 2013).

3.3. Condensed tannin content

The determination of condensed tannin content was done using the vanillin-HCl assay, which is based on the measurement of red color result when the vanillin reacts with meta-substituted ring of flavonols. However, the fact that any substituted monomeric flavanol can react with the vanillin makes this assay nonspecific for condensed tannin determination (Schofield et al., 2001). The results of condensed tannin content which showed significant differences among analysed date fruit varieties was illustrated in Table1. Bousrdon cultivar showed the highest level of condensed tannins (92.141 mg CE/100 g DW) while the lowest level was observed in Bouskri cultivar (57.564 mg CE/100 g DW). Our results are lower than those reported by Benmeddour et al, (2013) who found that condensed tannin content varied between 82.81 and

525.06 mg CE/100 g DW.

con pos

3.4. Antioxidant activities

Date fruit contains a wide range of phenolic compounds, which have diverse antioxidant capacities. To better examine their antioxidant capacities, different assays are required. Hence, in this study the ferric reducing ability (FRAP) and free radical scavenging activity assay DPPH were used in this respect. Simplicity, rapidity, reproducibility as well as the excellent relationship between the molar concentration of antioxidant and the ferric reducing ability, make FRAP the better assay to assess the antioxidant potential of food polyphenols compound (Müller et al., 2010). This method is based on the ability of electron-donating antioxidants to reduce at an acidic medium, a colorless ferric complex (Fe3+-tripyridyltriazine) to blue-colored ferrous complex (Fe2+-tripyridyltriazine) which had a maximum absorbance at 593 nm (Dudonne et al., 2009). The result showed that all cultivars exhibited a good reducing power which is varied significantly (P < 0,05) from 406.61 to 860 ¡irnol TE/100 g DW for Jihl and Bouskri, respectively. As shown in this study (Table 4), there is a strong positive relationship between the antioxidant activity measured by FRAP assay and the total polyphenol contents (r = 0.871), flavonoids content (r = 0.948), as well as condensed tannins (r = 0.952). Therefore, flavonoids are the principal provider to the antioxidant potential of date fruit extract. DPPH (1,1-diphenyl-2-picrylhydrazyl) assay is one of the well-known and widely used method for estimating antioxidant activity which is easy to perform and has an acceptable accuracy (Zhou and Yu, 2004). DPPH is a stable free radical, when it accepts an hydrogen radical or an electron from an antioxidant compound it becomes a stable diamagnetic molecule and loses its purple coloration which measured at 517 nm.

The averages of free radical scavenging activity of date fruit cultivars using DPPH assay are displayed in Table 1, which showed a significant difference between examined date fruit varieties. Among these varieties Jihl cultivar (2.046 g of date/L ) exhibited the highest level of scavenging activity based on DPPH assay with a lower IC50 value (IC50 value is the concentration of the sample required to inhibit 50 % of radical) while Bouskri cultivar possessed the lowest level of antioxidant activity with a high IC50 (6.255 g of date/L). The scavenging activity of date fruit is still very low compared to the synthetic antioxidant (Trolox) which possessed an DPPH IC50 (0.0101 g of Trolox /L).

The analysis of correlation (Table 4) between phenolic, flavonoid and scavenging activity indicated a strong negative correlation. phenolic content/DPPH (r = -0.90) and DPPH/flavonoid content (r = - 0.842) as well as DPPH/condensed tannins (r = - 0.942). These

strong negative correlations suggest that the phenolic compounds, especially flavonoid may be the main provider of the scavenging ability observed on date fruit.

3.5. Antihemolytic activity

The high membrane concentrations of polyunsaturated fatty acids (PUFA) and the hemoglobin redox reactions associated with O2 transport, which are the potent promoters of reactive oxygen species make the Erythrocytes the first targets for free radical attack (Nabavi et al., 2011), hence the need for their membrane protection. Table 2 illustrated the protective effect of date fruit extract on erythrocytes treated with a free radical generator (AAPH), which enhance membrane lipid peroxidation a key factor for cell lysis. Among analyzed date fruit varieties, Jihl exhibited considerable antihemolytic activity with a half time of hemolysis (210.99 min) more than the control (Trolox) with a half time of hemolysis (175.84 min), while Bouskri exhibited the lowest activity with a half time of hemolysis (158.70 min). The high positive correlation as showed the Table 4 between flavonoid content and half time of hemolysis (r = 0.887) in one hand, and phenolic content and the increase on half time of hemolysis (r = 0.690) in other hand, justified the involvement of flavonoids and other polyphenols on the erythrocyte membrane stability as mentioned by various authors (De Freitas et al., 2008; Chaudhuri et al., 2007). Scavenging of lipid peroxyl radicals generated by AAPH seems to play a considerable part in antihemolytic activities as showed a high correlation between antioxidant assays and antihemolytic activity (Table 4). The nonsignificant hemolysis observed when erythrocytes were treated only with date fruit extract as showed the Table 3 can justified that these extract are nontoxic and harmless for the cells. However it possessed a membrane stabilizing effects varied between 33.84 % for Jihl and 0.44 % for Bouskri compared to the control.

3.6. Antibacterial activity

The antibacterial screening of six Moroccan dates fruit cultivars (Bousrdon, Jihl, Boufgous, Bousthammi, Majhoul and Bouskri) against S. aureus, B. cereus, B. subtilis, P. aeruginosa, E. coli, S. abony was shown in the Table 5. The antimicrobial activity of the samples was determined using the minimal inhibitory concentration (MIC) values for the tested bacteria. All the tested date fruit extracts shown an antibacterial activity except Majhoul and Bouskri extracts but this antibacterial effect still lower compared to the gentamicin ( 10 ^g/disc) used as reference antibiotic. This effect is varied between varieties and depends also on the tested bacterial strains.

Bousrdoun extract showed a higher antibacterial activity among analysed varieties. The highest antibacterial activity of Bousrdoun was recorded against S. abony and against B.

cereus with a MIC value 2.5 mg mL-1, the same antibacterial activity against B.cereus was shown using Jihl extract. The Majhoul and Bouskri varieties showed no activity against all bacterial strains tested.

The difference in the sensitivity of studied bacterial strain against date fruit extract may be due to the change on their inherent structural or functional characteristics, which allow them to tolerate of date fruit compounds. The nature and the combination of polyphenolic compounds present in the date fruits extract may also be critical in this respect. Several studies attributed the inhibitory effect of plant extracts against pathogenic bacteria to their polyphenolic compounds, precisely the ability of phenolic compounds to bind with the bacterial cell wall and then inhibiting the bacterial growth (Sellam et al., 2013; Barbary et al., 2010; Baydar et al., 2004). These phenolics compounds may act through their ability to precipitate protein and inhibit enzymes of microorganisms (Naz et al., 2007). The results showed a good association between the concentrations of polyphenols compounds and the antimicrobial activity. 4. Conclusion:

The study revealed that compounds present in date fruit exhibit antioxidant, antihemolytic and antibacterial activities. The high correlation between these biological activities and antioxidant property has focused our attention on the role of phenolic as the main responsible factor of these biological activities due to their antioxidant power.

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Antioxidant activity, total phenolic content, total flavonoid and condensed tannin content of different date varieties from Morocco.

Total Phenolic content mg GAE/100g DW Total Flavonoid content mg RE/100gDW Condensed Tannins content mg CE/100gDW FRAP l^mol TE/100g DW DPPH g of date/L.

Boufgous 506.77+ 23.95a 84.35+2.95a 81.34 +2.64 627.99+27.59 3.42+0.08

Bouskri 331.86+13.24 68.87+2.14 57.56 +3.86 406.614+14.31 6.25+0.13

Bousrdon 537.074+19.25 188.58+4.45 92.14 +4.68 818.86+21.91 3.11+0.08

Bousthammi 441.851+29.79 85.07+100 a 69.90 +3.97 530.23+15.90 4.79+0.12a

Jihl 495.269+20.50a 208.53+4.51 87.07 + 5.29 860.89+17.08 2.05+0.03

Majhoul 398.228+21.58 77.73+3.65 64.29 + 5.7 469.03+17.99 5.25+0.19a

Trolox 0.0101+0.0019

Values in average (n=6) ± SE. Averages, in the same column, with different letters are significant

nt using post hoc Bonferroni tests (p < 0.001)

Table 2

Antihemolytic activity of different date fruit extract.

Hemolysis half-time (min) Protective effect %

Control 175,84 + 4.04 e f -

AAPH-blood 85.66 + 3.46 -

AAPH-blood-Bou/gous extract 176.90 + 2.44 a b 206.51%

AAPH-blood-Bous&ri extract 158.70 + 2.10 185.27%

AAPH-blood-Bousrdon extract 184.49 + 3.62 a c 215.37 %

AAPH-blood-Bousthammi extract 177.44 + 2.59 c' d' e 207.14 %

AAPH-blood-Majhoul extract 170.37+ 4.67 b' d' f 198.90 %

AAPH-blood-/ihl extract 210.99 + 3.11 246.31 %

AAPH-Trolox 1% 182.17 + 5.29 212.67 %

Values in average (n=6) + SE. Averages, in the same column, with differe using post hoc Bonferroni tests (p < 0.001)

Table 3

Evaluation of hymolysis and Membrane stabilising

etters are significantly different

induced by date fruit extract

Hemolysis half-time (min) Membrane stabilising effects %

Control 175,84 + 4.04

Blood-Boufgous extract 184.82 + 1.47 5.11 %

Blood-Bouskri extract 176.62 + 1.57 0.44 %

Blood-Bousrdon extract 195.03 + 2.60 10.91 %

Blood-Bousthammi extract 186.34 + 2.10 5.97 %

Blood-Majhoul extract 179.07 + 3.37 1.84 %

Blood-/ihl extract 235.34 + 3.17 33.84 %

Values in average (n=6) + SE. Averages, in the same column, with different letters are significantly different using post hoc Bonferroni tests (p < 0.001)

Correlation phenolic, flavonoid and condensed tannins content with antioxidant and antihemolytic activities.

Polyphenols Flavonoids CT FRAP DPPH AhE

Polyphenols 1

Flavonoids 0.688 1 S

CT 0.967 0.836 1 G-

FRAP 0.871 0.948 0.960 1 à

DPPH -0.900 -0.842 -0.942 -0.960 1 J

AhE 0.690 0.867 0.770 0.890 -0-912

CT: Condensed Tannins; AhE: Antihemolytic effect

Antibacterial activity of six date fruit varieties against the bacterial strains based on disc diffusion method

Bousrdoun Jihl Boufgous Bousthammi Majhoul Bouskri Gentamicin

Bacterial strains

Gram-negative bacteria

P.a ATCC 27853 12.33+0.44 11.66+0.44 10.66+0.44 9.00+0.66 Na Na 17.66+0.33

E. c ATCC 25922 12.33+0.44 10.66+0.44 10.66+0.44 8.00+0.66 Na Na 19.00+0.00

ab NCTC 6017 14.66+0.44 11.66+0.66 12.00+0.00 10.00+0.00 Na 4 Na 18.66+0.22

Gram-positive bacteria

B. c ATCC 29213 14.00+0.00 12.00+0.00 12.33+0.44 10.00+0.00 Na Na 20.33+0.22

B. s ATCC 6633 12.66+0.44 11.66+0.44 11.00+0.00 9.33+0.44 Na Na 19.66+0.33

S. a ATCC 25923 13.66+0.44 11.66+0.44 11.00+0.66 7,66+0,44 Na Na 18.00+0.00

P.a: Pseudomonas aeruginosa; E.c: Escherichia coli; S.ab: Salmonella abony; S.a: Staphylococcus aureus; B.c: Bacillus cereus; B.s: Bacillus subtilis. Na: Not active.

ms; B.c: Ba

The MIC values of six date fruit varieties against the Bacterial strains tested

Minimum Inhibitory Concentration (MIC) mg.mt1 A

Bousrdoun Jihl Boufgous Bousthammi Majhoul Bouskri

Bacterial strains V

Gram-negative bacteria

P.a ATCC 27853 5.00 5.00 10.00 10.00 Nt Nt

E. c ATCC 25922 5.00 10.00 10.00 >10.00 Nt Nt

ab NCTC 6017 2.50 5.00 5.00 10.00 Nt Nt

Gram-positive bacteria

5. a ATCC 25923 5.00 5.00 10.00 >10.00 Nt Nt

B. c ATCC 29213 2.50 2.50 5.00 10.00 Nt Nt

B. s ATCC 6633 5.00 5.00 10.00 >10.00 Nt Nt

P.a: Pseudomonas aeruginosa; E.c: Escherichia coli; S.ab: Salmonella abony; S.a: Staphylococcus aureus; B.s: bacillus subtilis; B.c: Bacillus cereus; Nt: Not tested.