INTRODUCTION:

Microbial contamination and side effects of synthetic antioxidants are two important major concerns of food and pharmaceutical industries. Increasing propensity for replacing synthetic antioxidant by natural one on one side and development of microbial resistance to existing antibiotics from the other has encouraged researchers toward appraising medicinal plants for dual antioxidant and antimicrobial properties.

Though, since immemorial time, medicinal plants have been used to treat and prevent various human ailments and they are considered as reservoir of bioactive compounds¹. Epidemiological studies have shown that a correlation exists between an increased consumption of phenolic antioxidants and a reduced risk of cardiovascular disease. The same relationship was also observed with certain types of cancer².

Peaches (Prunus persica L). are nutritionally and economically important and they are one of the most popular fruits consumed worldwide. Belongs to the family Rosaceae is a deciduous tree with a height of 5 to 10 m and is commonly cultivated in West Asia, Europe, India and North Africa³. There are about 100 genera and 3,000 species in Rosaceae family⁴. P. persica L. leaves are astringent, anthelmintic, insecticidal, vermicidal, demulcent, diuretic, expectorant, febrifuge, laxative, parasiticide, mildly sedative and are used in leucoderma and in piles. They are used internally in the treatment of gastritis, whooping cough, coughs and bronchitis⁵. Leaf paste is used to kill worms in wounds and fungal infections⁶.

Till now there is limited data in research focus on the effect of Saharian area in the cultivation of fruit in Algeria, the above mentioned literature survey have prompted us to undertake the present study to assess the effect of the semi-arid climate on the bioactive compounds of peach leaves used in the recipes of many type of cancers and tumors in Bechar region, by determining the phenolic contents as well as to evaluate in vitro the biological activities of the differents extracts using various models, and to investigate their kinetic behaviour of DPPH radical scavenging activity.

MATERIALS AND METHODS:

Plant material:

The leaves of P. persica L. were harvested in March 2016 from Bechar, Algeria. The plant collected wasidentified in the National Conservation Agency Bechar Unit, Algeria. Plant samples were dried in the share and conserved until use.

Extraction of chemical compounds:

The leaves 40g in 300mL were subjected to two types of extraction decoction with methanol, ethanol and distilled water at 60˚C during 3 hours and maceration with acetone, ethyl acetate and chloroform with stirring for 48 hours, in the shade and at room temperature. The extracts recovered by filtration is subjected to evaporation of the solvent under reduced pressure in a rotary evaporator (Buchi), and stored at 4°C until it is used.

Determination of total phenolic contents:

Total phenolic content in all extracts was determined using Folin-Ciocalteu reagent method⁷.Briefly, 200μL of plant extract mixed thoroughly with 1mL of Folin-Ciocalteu reagent diluted 10 times. After 5 min, 0.8mL of 7.5% Na₂CO₃ solution was added to the mixture. This later was allowed to stand in darkness for 30 min at 25°C. The absorbance was determined using spectrophotometer (UV-VIS 1700 pharma spec SHIMADZU) at 765nm. The total phenolic content was expressed as mg GAE/g DW.

Determination of total flavonoid contents:

Total flavonoid content was quantified according to a standard method using quercetin as a standard.The plant extract of 500µL was added to 1.5mL distilled water followed by 150µL of 5% NaNO₂ solution. After 5min of incubation at 25°C, 150µL of 10% AlCl₃ was added. After another 6min, the reaction mixture was treated with 0.5mL 1M NaOH. Then the absorbance was measured at 510nm. The flavonoid content was calculated from a quercetin curve expressed as mg QRE/gDW^8,9.

Determination of total condensed tannins:

The analysis of condensed tannins was carried out according to the method of Julkunen-Titto¹⁰. To 400μL of properly diluted sample; 3mL of 4% vanillin solution in methanol and 1.5mL of concentrated hydrochloric acid were added. The mixture was allowed to stand for 15 min, and the absorption was measured at 500. The amount of total condensed tannins is expressed as mg CAT/g DW.

Determination of total flavonols:

Total flavonols in the plant extracts were estimated using the method of Almaraz-Abarca^11,12,13. To 1mL of sample, 1mL of 2% AlCl₃ in ethanol and 3mL sodium acetate (50g/L) solutions were added. The absorption at 440nm was read after 2.5 hours at 25°C. Total content of flavonols was expressed in terms of quercetin equivalent, mg QRE/g.

Antioxidant activity:

Thin layer chromatography antioxidant assay:

The extracts were subjected to TLC qualitative antioxidant assay on a silica gel plate (20×20cm, silica gel F254, Merck). The extracts were loaded on CCM plate and were sprayed with 0.04% (w/v) DPPH reagent prepared in methanol. The spots were observed after they had been heated at 60°C for 30 min^14,15,16.

Free radical scavenging activity, DPPH assay:

The free radical scavenging activity was measured by a modified DPPH^•assay. Briefly, 100μL of the extract was added to 1.9mL of DPPH solution (0.04%). The mixture was vigorously shaken and then allowed to stand 30 min in the dark. The absorbance of the mixture was measured at 517nm. The scavenging activity was expressed as inhibition percentage using the following equation:

A_c– A_s

DPPH* (%) = --------------- X 100

A_c

A_c: is the absorbance of control reaction

A_s: is the absorbance of sample solution containing the test compound.

The ascorbic acid and BHT were used as a positive control. The extract concentration providing 50% inhibition (IC₅₀) was calculated from the graph of inhibition percentage plotted against extract concentrations¹⁷.

Kinetic analysis:

To follow the kinetic 1.5mL of extract was mixed with 1.5mL of DPPH solution (0.04%), the absorbance was measured at 517nm after each 30 seconds until it becomes constant, results compared with ascorbic acid and BHT. The DPPH^·concentration in the reaction medium was calculated from the following calibration curve, determined by linear regression:

A517 nm = 17.531 × [DPPH*] t + 0.039 R² = 0.999

Where: [DPPH^·]t was expressed as mg/ml at t time. The percentage of the remaining DPPH^·(% DPPH^·_REM) at the steady state was calculated as follows:

Where: [DPPH^·]t=0 and [DPPH^·]t are concentrations of DPPH^·at t=0 and t= t, respectively.

Using various antioxidant concentrations, it was possible to determine the amount of antioxidant necessary to halve the initial DPPH^·concentration (EC₅₀). It is expressed in mg of dry extract per g of DPPH. The time needed to reach the EC₅₀concentration, noted TEC₅₀was graphically determined.The antiradical efficiency (AE) was calculated as follows:

Authors have classified the kinetic behavior of the antioxidant as follows: fast (TEC₅₀< 30 min), medium (TEC₅₀: 30-60 min) and slow reaction kinetics (TEC₅₀> 60 min)¹⁸. According to Sanchez-Moreno etal.¹⁷the following classification of antiradical efficiency was proposed: ARE < 1×10^-3 slow, [1×10^-3; 5×10^-3] intermediate, [5×10^-3; 10×10^-3] high, and ARE > 10×10^-3 very high.

Reducing power capacity:

According to the method of Oyaizu¹⁹, various amounts of the extracts were mixed with phosphate buffer (2.5 mL, 0.2 M, pH 6.6) and 2.5mL of potassium ferricyanide 1% solution. The mixture was incubated at 50°C for 20 min. 2.5mL of trichloracetic acid 10% were added to the mixture which was then centrifuged for 10 min. The supernatant 2.5mL was mixed with 2.5mL of distilled water and 0.5mL of a freshly prepared FeCl₃ 0.1% solution. The absorbance was measured at 700nm. Ascorbic acid and BHT were used as a positive control. In this method, increased absorbance indicates an increased reducing power.

Phosphomolybdenum reduction assay:

The assay is based on the reduction of Mo (VI) to Mo (V) by the extracts and subsequent formation of green phosphate/Mo (V) complex at acid pH. 0.3mL of extract was combined with 3mL of reagent solution (0.6M sulfuric acid, 28mM sodium phosphate and 4mM ammonium molybdate) and incubated at 95°C for 90 min. The absorbance was measured at 695nm. The total antioxidant capacity is expressed in milligram equivalents of ascorbic acid per gram of the dry weight (mg EAA/g DW)^20,21,22.

Hydrogen peroxide scavenging activity:

1 mL of extract was mixed with 2mL of 40mM H₂O₂ solution prepared in phosphate buffer (0.1M pH 7.4) and incubated for 10 min. The absorbance was taken at 230nm²³. The amount of hydrogen peroxide radical inhibited by the extract was calculated using the following equation:

Where: A_c: is the absorbance of control reaction of H₂O₂ radical + methanol

A_s: is the absorbance of sample solution containing the test compoundor standard.

FRAP assay:

The stock solutions included 300 mM acetate buffer (3.1 g C₂H₃NaO₂.3H₂O and 16mL C₂H₄O₂), pH 3.6, 10mM 2, 4, 6- tripyridyl-s-triazine (TPTZ) solution in 40 mMHCl, and 20mM FeCl₃.6H₂O solution. The fresh working solution was prepared by mixing 25mL acetate buffer, 2.5mL TPTZ, and 2.5mL FeCl₃.6H₂O. Extract 200μL was allowed to react with 2800μL of the FRAP solution for 30 min in the dark condition. Readings of the colored product (ferrous tripyridyltriazine complex) were taken at 593nm^24,25,26. The standard curve of FeSO₄was linear. Results are expressed in mg Fe (II)/g dry weight and compared with ascorbic acid and BHT.

ABTS radical cationdecolorization assay:

The ABTS radical was generated during a chemical reaction between the 7mM aqueous solution of ABTS diammonium salt and the 2.45 mM potassium persulfate. The solution was kept at a room temperature in darkness throughout the night, in order to complete the reaction and to stabilize the ABTS cation-radical. Prior to analysis, the radical solution was diluted with ethanolto obtain final absorbance value of A = 0.70±0.02 measured at 734nm. 200μL aliquots of the properly diluted extract or of standards solutionswere added to 2 mL of ABTS 0.7 and absorbance value was measured 6 min after mixing. The percentage of inhibition of ABTS^·⁺ was calculated using the formula as DPPH method^27,28.

Antimicrobial activity:

Strains tested:

Salmonella typhimurium ATCC13311, Bacillus cereus ATCC 213332, Listeria monocytogenes ATCC 15313 and Staphylococcus aureus ATCC 25923 were from the Vet Agro Sup, Veterinary School of Lyon, France. Candida albican ATCC 10231, Candida albican ATCC 26790 and Candida albican CIP444 were performed by the Laboratory of biology, Tlemcen (Algeria). Six pathogenic strains of fungi were used in the study, namely, Aspergillus parasiticus, CBS 100926T, Biocentrum-DTU Microbial Biotechnology Center, Technical University Denmark, Aspergillus alliaceus, isolated local wheat durum variety Waha from the region of Setif and molecularly identified by sequencing IT1-ITS2 regions and calmodulin, and four others Fusarium graminearum, Fusarium oxysporum, Pencillium expansum and Pencillium glandicola, isolated from wheat, were provided by VRVSSAA Research Laboratory of Tahri Mohamed University of Bechar, Algeria. Strains tested were stored in the appropriate medium before use and conserved at 4°C.

Evaluation of the antifungal activity:

The antifungal activity was determined by using the agar well diffusion method, 1 mL of extracts tested (12.5-0.78 mg/mL), were added to 19 mL of a solution of sterilized potato dextrose agar acidified. The mixtures were cast on the Petri dish. Thereafter, amycelial disc of 6 mm in diameter, cut from the periphery of new culture in agar 2% of 16 h, was inoculated in the centre of each Petri dish, and then incubated at 25±2°C. The diameters of growth of the hypha were recorded after 7 days²⁹. The antifungal index was determined using the following formula:

Where: D_a and D_b are the diameter of the growth zone in test and in control Petri dish (mm).

Evaluation of the antibacterial activity:

The disc diffusion method was used for the determination of the antibacterial activity. Sterile discs impregnated with 20μL of extracts tested, were placed in Petri dishes on Mueller-Hinton agar, which had been surface spread with 1mL of bacterial solution adjusted to a 10⁸ UFC/mL fixed by the optical density (0.080.1 at 620nm)³⁰. The Petri dishes were then incubated for 18 h at 37°C. The diameter of the inhibition zone was measured.The results of the extracts were compared with a standards, positive control (Ampicillin 10µg, Gentamicin 10µg, Penicillin 10 µg, Chloramphenicol 30 µg), and negative control (solvent of extract DMSO 5%). The same procedure used for Candida strains using Sabouraud agar, adjusted strains to (0.12-0.15) at 530 nm which is equivalent to 10⁷ UFC/mL. The Petri dishes were then incubated for 48 h to 72 h at 37°C.

Determination of minimum inhibitory concentration (MIC):

Various concentrations of extracts between 12.50.012 mg/mL were introduced into different test tubes; each tube was inoculated with an overnight culture of S. aureus, B. cereus, L. monocytogene and S. typhimurium diluted to give a final concentration of 10⁶ cells per mL. The tubes were incubated at 37°C for 24 h. 2 tubes were used as control. The concentration of the extract that did not permit any visible growth of the inoculated test organism in broth culture was regarded as the minimum inhibitory concentration (MIC) in each case³¹.

Determination of minimum bactericidal concentration (MBC):

After culturing the test organisms separately in nutrient broth containing various concentrations of active ingredients, the broth was inoculated onto freshly prepared agar plates to assay for the bactericidal effect. The culture was incubated at 37°C for 24 h. The lowest concentration of extract tested that does not yield any colony growth on the solid medium after the incubation period was regarded as minimum bactericidal concentration (MBC)³².

Statistical analysis:

The experimental results were expressed as mean ± standard deviation (SD) of 3 replicates. Where applicable, the data were subjected to one way analysis of variance (ANOVA) and differences between samples were determined by Duncan’s Multiple Range test using the Graph Pad Prism software version 5.03 program. P Value < 0.05 was regarded as significant. Correlations between the parameters evaluated were obtained using Pearson’s correlation coefficient (r).

RESULTS AND DISCUSSION:

Yield and total phenolic contents:

The results showed that leavesextraction yield of P. persica L. varied considerably as a function of solvent nature and ranged from 3.57 to 33.77% (Table 1). Extraction with water gives the highest amount of total extractable compounds, whereas chloroform yield was small in comparison to other solvents. The yields of decocted extracts were more important than macerated. These results may be compared with a previous report³³, which indicated maximum yield was obtained in methanol fraction (16.3±0.4%). The extraction yield depends on solvents, time, and temperature of extraction as well as the chemical nature of the sample³⁴.

Different amount of polyphenols were obtained. Generally, the chloroformic extract exhibited the highest total concentration of total phenolic, flavonoids, tannins and flavonols contents. While the lowest amount of the flavonoids, tannins and flavonols contents were observed in the aqueous extract (Table 1). Previous report³⁵, indicated that hydromethanolic extract of the leaves of P. persica L. contained 19.3±1.1mg GAE/g DW. It is strongly believed that whenever the molecular weight of the solvent is higher, the polarity is lower, which allows to easily extract other substances having the same molecular weight. This can be associated to ‘‘like dissolves like” or ‘‘polarity versus polarity” principle as both chloroform and tannins are of high molecular weight. This solvent has the lowest polarity but contains the highest total phenolic compounds value³⁶.

Thin layer chromatography antioxidant assay:

In this study, TLC bioautography was used to detect the phenolic and antioxidant substances in P. persica L. extracts. Different yellow spots were visualized after spraying with 0.004% DPPH reagent; this antioxidant activity observed contribute to the presence of several bioactive compounds in the leaves extracts tested. Our results are in accordance with those reported by Molyneux³⁷.

DPPH free radical scavenging activity:

Six extractives exhibited considerable DPPH free radical scavenging activity as indicated by their IC₅₀ values. Standards ascorbic acid and BHT were found to have an IC₅₀0.04±0.01 and 0.44±0.04mg/mL, respectively. In comparison to ethanolic, aqueous and methanolic extract displayed IC₅₀ of 1.03±0.10mg/mL, 1.19±0.06mg/mL and 1.34±0.20mg/mL, respectively. The chloroformic extract is seen to have the least free radical scavenging activity 2.73±0.17mg/mL.Nevertheless, when compared to standards, all the tested extracts showed statistically significant (p < 0.05) in the DPPH radical scavenging activity (Figure 1).The outcomes of the study are in good agreement with the previous findings³⁸, where P. persica L. exhibited moderate DPPH radical scavenging activity ranged between (38.10-48.08%) in the methanolic, aqueous and acetonic extracts. The difference in the DPPH radical scavenging activity in different solvent extracts implies towards the preference of the solvents for extraction of different types and concentrations of bioactive compounds^39-40.

Figure 1. DPPH radical scavenging activity of different fractions of P. persica L. (I%) at different concentrations. Aq, Aqueous; Metha, Methanolic; Etha, Ethanolic; Etac, Ethyl acetate; Aceto, Acetonic and Chlor, Chloroformic

Kinetic analysis of DPPH activity:

The reduction in the absorbance of the DPPH^•radical, which corresponds to the reduction in the percentage of DPPH^•remaining at the stationary phase, was explained by its reduction in the presence of the extracts or controls as function of time (Figure 2). The percentages of [DPPH]_REM of decocted extracts aqueous, ethanolic and methanolic at 12500mg of dry extract per g DPPH were 4.71 and 6.59 and 9.02% respectively, and those for the macerated extracts were 14.22, 20.17 and 36.03% respectively for acetonic, ethyl acetate and chloroformic.

The EC₅₀ and antiradical efficiency (AE) values of scavenging DPPH radicals of all extracts ranged from 213.55 to 502.48 mg of antioxidant/g DPPH^•and from 3.62 × 10^-4 to 2.20 × 10^-4mg/g.min, respectively. Taking the classification standards described above as a reference, it is evident that the chloroformic extract react rapidly with the DPPH radical better than standard of the ascorbic acid, while the BHT, was included in the low effectiveness category. All the brut extracts tested were considered as reacting at fast speed. From above experiment, it can be mentioned that different solvents extractives of P. persica L. leaves may possess significant effect on kinetic study of DPPH free radical scavenging activity.

Figure 2. Kinetic behavior of different fractions of leaves of P. persica L. at different concentrations. Aq, Aqueous; Metha, Methanolic; Etha, Ethanolic; Etac, Ethyl acetate; Aceto, Acetonic and Chlor, Chloroformic

Phosphomolybdenum assay:

The formation of the complex was measured by the intensity of absorbance in extracts at concentration of 0.5mg/mL. All the test samples exerted noticeable antioxidant activities that ranged from 107.29±18.39 to 501.88±31.49mg AAE/g DW in six different extracts tested. The highest antioxidant activity was observed in chloroformic extract 501.88±31.49mg AAE/g DW, while the aqueous extract was found 107.29±18.39mg AAE/g DW as lower one, in comparison to BHT (846.54±108.56mg AAE/g DW). The extracts tested were varied significantly among each other (P< 0.05). However, in comparison to another study⁴¹the total antioxidant in the present investigation was found to be much higher.

Reducing power assay:

Figure 3 shows that P. persica L. extracts had effective reducing powerusing the potassium ferricyanide reduction method when comparedto the standards (Ascorbic acid and BHT). Thereducing power increased steadily with increasing concentration of samples. Methanolic extract showed the highest reducing power (IC₅₀ 0.521±0.02mg/mL), while both aqueous (0.967±0.13mg/mL)and ethyl acetate (0.929±0.13 mg/mL) extracts were found to be nearer to each other. However the chloroformic extract was the lower, these differences were statistically very significant (p<0.05). By comparing these findings with that reported by Dhingra et al.⁴², we observed that the reducing power of ethyl acetate fraction obtained from whole fruit were higher than the leaves fraction with the same solvent showing a reducing power of IC₅₀ value of 0.112±0.00 mg/mL. The reducing capacity of compounds serves as an important indicator of their potential antioxidant activity⁴³.

Figure 3. Reducing power of extracts from leaves of P. persica L. at different concentrations. Aq, Aqueous; Metha, Methanolic; Etha, Ethanolic; Etac, Ethyl acetate; Aceto, Acetonic and Chlor, Chloroformic

Hydrogen peroxide scavenging activity:

Scavenging of hydrogen peroxide of various extracts indicated that ethanolic extract exhibited maximum inhibition 31.53±0.37%. In addition, all of aqueous, methanolic and BHT showed identical activity in depleting H₂O₂ with 29%, while acetonic and chloroformique with 26%, but the results remain lower than those of ascorbic acid 33.06±1.02%, (P< 0.05). Previous studies have suggested that P. persica L. showed 60-70% of scavenging activity⁴⁴.

ABTS radical scavenging activity:

All the extracts tested scavenged ABTS radical in a concentration-dependent way. Significant difference (p <0.05) was revealed between ABTS scavenging capacities of extracts measured. Present results showed that the ability of samples can be ranked as ethanolic> aqueous >methanolic>chloroformic>acetonic> and ethyl acetate which were exhibited prominent ABTS radical scavenging activities. The decocted extracts were fast and effective scavengers of the radical than macerated one and this activity was comparable to that of ascorbic acid and BHT. At 0.5mg/mL the percentage inhibition of decocted extracts were 76.30±4.35%, 73.15±7.81% and 71.30±7.95% for ethanolic, methanolic and aqueous extracts, respectively. While the macerated extracts the percentage inhibition does not exceed 49.97%. On the other hand, ascorbic acid and BHT exhibited respectively 94.47±0.02%, 94.46±0.16%. These results suggest a probable paramount role that the polyphenolic constituents of the extracts might play in free radical neutralization.

FRAP assay:

Antioxidants can be explained as reductants, and inactivators of oxidants⁴⁵. The results of the FRAP assay reported that in the decocted extracts, maximum amount was obtained in ethanolic extract 253.3mg Fe(II)E/g, whereas the minimum value of 60mg Fe(II)E/g dry plant was observed in aqueous extract. However, the macerated extracts showed a considerable antioxidant effect ranged from 106.66 to 163.33mg Fe(II)E/g, Furthermore the BHT activity was found 266.66 mg Fe(II)E/g, (P< 0.05). All the samples tested possessed different antioxidant activity power.Halvorsenet al.⁴⁶, suggested most of the secondary metabolites are redox-active compounds that will be picked up by the FRAP assay.

Correlation different antioxidant methods with phenolic contents:

In order to more appreciate the relationships between antioxidant capacities and phenolic content of leaves extracts of peach, correlations between assays under different extracting conditions were analyzed. Under the parameter of solvent type, the correlations between TPC, TFC and antioxidant assays (DPPH-RSA, ABTS and reducing power) were positively high (0.50 < R < 0.91, P< 0.05). Similarly, a strong correlation was also drawn between condensed tannins was observed just in DPPH, ABTS assays with (0.84< R < 0.93, P < 0.05), in contrast there was a weak correlation between tannins and flavonols with reducing power assay. The significant correlation supports and confirms that a potent antioxidant activity might be linked to the important amount of polyphenols in extracts in the current study. We can suggest that the hydrogen electron donating abilities of leaves extracts were directly proportional to the concentration of total phenolics. The antioxidant activity of medicinal plants, fruits and vegetables has been reported to be positively correlated to their total phenolic contents due to their ability to scavenge free radicals⁴⁷.

Antibacterial activity:

The aqueous extract showed maximum inhibitory activity against S. aureus 12±01mm at 1.56 mg/mL, followed by B. cereus 10±0.5mm and S. typhimurium 10.16±0.76mm at 6.25 mg/mL, however weak activity against L. monocytogenes. While the most interesting effect of the ethanolic extract was against L. monocytogenes at 12.5mg/mL. Whereas a resistance of S. aureus and S. typhimurium towards the ethanolic and methanolic extracts was observed. In addition, the same activity of the ethanolic extract was remarked against C. albican ATTC 10231 at 12.5mg/mL, but the aqueous and methanolic extracts have shown weak activity against C. albican ATTC 26790 and C. albican CIP444.

Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC)

From the results obtained of MIC, we noticed that the absence of turbidity was observed at the concentration 12.5mg/mL for the aqueous extract against S. aureus and B. cereusstrains, whereas methanolic and ethanolic extracts against S. aureus and B. cereus respectively. For the MBC, in solid medium, the total absence of colonies was observed at the same concentration. It is possible to deduce that the action of the extracts tested on the bacterial strains studied is bactericidal.

Antifungal activity:

All the extracts under study exerted an inhibitory activity on fungal strains mycelium growth. The ethanolic extract was the most potent; the high percentage of inhibition was 20.59±17.14% at 0.78 mg/mL against P. glandicola, similar effect against F. oxysporum at 1.56 mg/mL, however less activity against the other fungal strains. The high activity of the methanolic extract was obtained at 0.78 mg/mL (18.05±10.28%) against F. oxysporum and medium activity against P. expansum. Furthermore, moderate activity exerced by the aqueous extract against P. expansum and A. alliaceus.

The finding agrees with previously published results⁴⁸of P. persica L. leaf extracts that exhibited antimicrobial activity against a group of gram-positive and gram-negative bacteria. In addition, Previous reports have indicated that the aqueous alcoholic, methanolic and acetonic extract of P. persica L. are known to possess remarkable biological activities and therapeutic effects including antibacterial potential, antifungal strains such as Streptococcus pyogenes, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa, Candida albicans and Aspergillus niger, Yersinia pestis, Bacillus cereus, Listeria monocytogenes⁴⁹.

CONCLUSION:

This study indicated that different types of extraction method and solvent had a big influence on the antioxidant property of obtained extracts. Because multiple reaction characteristics and mechanisms are likely to be involved, no single assay will accurately reflect all antioxidants in a mixed or complex system. Thus, to fully elucidate a full profile of antioxidant capacity of different solvent extracts, different antioxidant capacity assays were used in this study. Additional studies are currently underway to assess the in vivo biological activities and to identify more specific phytochemicals responsible for their antioxidant and antimicrobic property.

ACKNOWLEDGMENTS:

We acknowledge the professor Angelic Kadjo from the veterinary School of Lyon France and the professor Yahia Chebloune Research Director, PAVAL Lab, department of Biology Grenoble, France for providing necessary bacterial strains.

CONFLICT OF INTEREST:

The authors declare that they have no conflict of interest.

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Received on 27.12.2021 Modified on 20.05.2022

Research J. Pharm. and Tech 2023; 16(1):354-362.

DOI: 10.52711/0974-360X.2023.00062

Extracts	Yields (%)	Total phenolic (mg GAE/g DW)	Total flavonoids (mg QRE/g DW)	Total tannins (mg CAE/g DW)	flavonols (mg QRE/g DW)
Aqueous	33.77±2.17	27.04±0.83	ND	2.45±2.40	3.4±0.26
Ethanolic	17.87±0.28	21.19±6.35	117.72±37.97	14.26±4.37	22.98±3.48
Methanolic	21.38±1.04	12.85±2.66	99.80±33.73	41.8±19.49	64.93±12.72
Ethylacetate	5.98±1.31	79.0±19.47	548.46±28.32	123.10±7.49	74.39±3.93
Acetonic	6.89±1.11	70.43±13.30	490.87±18.21	111.73±10.42	74.18±5.35
Chloroformic	3.57±0.78	116.83±4.31	637.78±46.01	141.89±5.63	91.32±4.36