Antioxidant and Biological Activities of Fresh and Dried Apricot Extracts Obtained by Cold Soaking and Ultrasonic Extraction

The objective of this study was to evaluate the antioxidant (AA), antibacterial, and antifungal activity of fresh and pre-treated apricot extracts, dried by microwave at different powers (200, 400, and 800 W), extracted by the cold soaking method, and ultrasound-assisted extraction (UAE). Biological activity (bacterial and fungal) was estimated by agar disk diffusion test against four bacterial strains ( Escherichia coli , Pseudomonas aeruginosa , Staphylococcus aureus and Streptococcus sp.), and two fungal strains ( Candida spp. and Geotrichum capitatum ). Methanolic extracts of apricot fruits: fresh, dried processed

1 Introduction described to control the myriad of degenerative diseases that influence human health. 5,7 These phytochemicals are the main contributors to the antioxidant, antimutagenic, antimicrobial, hepatoprotective, cardioprotective and anti-inflammatory properties of apricots, in addition to their role in taste, colour, and nutritional value. 8 Pathogenic microorganisms are the main cause of infectious diseases in the world and have been considered a major public health problem worldwide. Plant extracts have been considered as a source of new antimicrobial agents for the future. Phenolic compounds are known to have activity against a wide range of microorganisms. Microbial pathogens in food may cause spoilage and contribute to foodborne disease incidence, while the emergence of multidrug-resistant and disinfectant-resistant bacteria, such as Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa has increased rapidly, causing the increase in morbidity and mortality. 9 Natural products like apricot could be used as effective drugs to treat human diseases, with high efficiency against pathogens and negligible side effects. Fresh and dried fruits are valuable components of our daily diet and are rich sources of antioxidants. For this purpose, we used two efficient methods for the extraction of bioactive compounds (polyphenols and flavonoids), such as cold soaking and ultrasound-assisted extraction (UAE). Cold soaking consists of soaking plant materials in a container sealed with a solvent and leaving them to stand at room temperature for 24 h, with frequent agitation. 10 The UAE was considered to be a simple, efficient extraction method, saving time and reducing chemical consumption. 11,12 In our work, the drying efficiency of apricots treated with sucrose, NaCl, and sodium bisulphite solutions dried in microwaves was tested at different powers with AA by scavenging DPPH free radicals, and biological activity tested with four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp.), and two fungal strains (Candida spp. and Geotrichum capitatum), and the same for apricot dough dried in an electric oven at 60 °C. The extraction was based on two methods: cold soaking and UAE. To our knowledge, such work has never been done before.

Preparation of apricot extracts
The variety of apricots selected in our study was widespread (local variety; rosé de Manaa from the region of Batna, Algeria). The fruits were randomly selected from several clusters at different heights and orientations, harvested at full maturity (July), and stored in cold storage at 4 °C.
In this work, the efficiency of the domestic microwave drying process of apricot was tested by AA by scavenging DPPH free radicals, and the biological activity was tested with four bacterial strains (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp.), and two fungal strains (Candida spp. and Geotrichum capitatum), and the same for apricot dough dried in an electric oven at 60 °C and compared to the raw material (apricot without drying). For this purpose, three treatments were carried out with the following solutions, sucrose (60° Brix), NaCl (6 %), and sodium bisulphite (6 %), before the drying. Then, the drying process was started at powers of 200, 400, and 800 W; the drying process was to be stopped at a residual moisture content of 5 %. In addition, the drying time was set according to the time prediction model obtained by the artificial neural network (ANN) (200, 400, and 800 W for 540, 440, and 320 s, respectively), according to a previous study 1 . For this purpose, microwave power (W), total apricot weight (g), moisture content (%), dry matter content (%), and moisture ratio (MR) were calculated and fed into the ANN model to predict drying time. Finally, the extraction of polyphenols and flavonoids was carried out for each treatment and each power according to two techniques: cold soaking and UAE, it should be noted that the present work is a continuation of a previous work already published in 2021. 1

Cold soaking
Four grams of fresh apricot and dry products were ground and homogenised, then macerated in 400 ml of methanol for 24 h under continuous stirring in a shaker. After filtration through 0.45 µm filter paper, the filtrates were concentrated in a vacuum rotary evaporator at 80 rpm rotation and 35 °C. After concentrating, the residue was recovered with 10 ml of pure methanol, and stored at −18 °C.

Ultrasound-assisted extraction (UAE)
UAE was performed using an ultrasonic bath (Soniclean 220 V, 250 W, and 50 kHz, Soniclean Pty Ltd., Thebarton, Australia). Four grams of fresh apricots were considered, and the processed products were crushed and homogenised followed by maceration in 400 ml of methanol for 30 min in an ultrasonic bath at 45 °C. Filtration was then carried out before concentration in a vacuum rotary evaporator. Recovery of the residue was performed with 10 ml of pure methanol; it was then stored at −18 °C.

Evaluation of the antioxidant activity of fresh apricot
and dried products by the effect of the free radical DPPH Free radical scavenging activity was measured employing the stable free radical DPPH (C 18 H 12 N 5 O 6 ). The reduction of this radical is accompanied by its change from the characteristic purple colour of the DPPH solution to yellow. 13,14 This radical is an oxidant that can be reduced by the antioxidant (AH) according to the following reaction: 15 Antioxidant activity was evaluated using the trapping method with modifications, where 1,1-diphenyl-2-picrylhydrazyl (DPPH) was used as a relatively stable free radical. An amount of 50 µl of the sample (fresh apricot extract and the processed products) was added to 1.95 ml of DPPH methanolic solution (6 • 10 −5 M) and vortexed. The reduction of this radical is accompanied by its change from the characteristic purple colour of the DPPH solution to yellow. 16 The mixture was left in the dark for 30 min, and the absorbance was measured at 517 nm with the corresponding blank. The AA is expressed in % and determined by applying the following equation: % AA = [(Abs control -Abs sample) / Abs control] · 100 (2) where % AA is antioxidant activity (percentage of anti-free radical activity), Abs sample is absorbance of the sample after 30 min, and Abs control is absorbance of the negative control after. [17][18][19][20] 2.3 Evaluation of the antibacterial and antifungal activity of Prunus armeniaca L.
The antimicrobial activity of the extracts was determined by the agar diffusion method standardised by the National Committee for Clinical Laboratory Standards. 21,22 The following bacterial and fungal strains were tested: Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp., Candida spp. and Geotrichum capitatum were used to test the effect of the extracts: fresh apricot, dry samples.
The samples had the following treatments: • Sample 1: Apricot was subjected to treatment with sucrose and dried at 800 W. • Sample 2: Apricot was subjected to treatment with NaCl and dried at 800 W. • Sample 3: Apricot was subjected to treatment with sodium bisulphite and dried at 800 W. • Sample 4: Apricot was subjected to treatment with sucrose and dried at 400 W. • Sample 5: Apricot was subjected to treatment with NaCl and dried at 400 W. • Sample 6: Apricot was subjected to treatment with sodium bisulphite and dried at 400 W. • Sample 7: Apricot was subjected to treatment with sucrose and dried at 200 W. • Sample 8: Apricot was subjected to treatment with NaCl and dried at 200 W. • Sample 9: Apricot was treated with sodium bisulphite and dried at 200 W, and extracted from the apricot dough dried in an electric oven at 60 °C.
The fresh and dried extracts of Prunus armeniaca L. were dissolved in methanol to a final concentration of 1 g/100 ml. 23,24 Bacterial and fungal strains were spread on nutrient and malt agar, respectively, incubated at 37 °C for 24 h, and 28 °C for 3-5 days to optimise their growth for bacteria and fungi, respectively. After growth, a suspension was prepared with diluted distilled water and adjusted to a concentration of 0.5 McFarland (10 6 CFU/ml).
Bacterial suspensions were spread on the surface of Mueller-Hinton agar using a swab to ensure uniform inocula-tion. Inoculation was performed three times while rotating the plate approximately 60 after each application. The discs impregnated with 50 µl of extracts were deposited with forceps on the surface of the inoculated agar. Two antibiotics (cefoxitin for gram-negative bacteria and gentamicin for gram-positive bacteria) were used as the positive, and the negative control was a methanol-impregnated disc. After 3 min of diffusion at laboratory temperature, the Petri dishes were incubated at 37 °C for 18 to 24 h, and the diameters of the inhibition zones were measured and discussed. The experiment was performed in duplicate. 25,26 The antifungal activity of the extracts was determined by the agar diffusion method. The stock cultures were grown on a suitable agar (Sabouraud with chloramphenicol and actidione), in order to inhibit the growth of the contaminating bacterial flora and to reach the stationary phase of growth. The discs impregnated with 50 µl of extracts were deposited with forceps on the surface of the Sabouraud agar. The fungi were incubated for 48 h at 30 °C. Antifungal activity was determined by measuring the diameter of the zone of inhibition.
Antimicrobial and antifungal activity was expressed by the diameter of the zone of inhibition of the strains against the extract, and interpreted according to Celikel  3 Results and discussion

Antioxidant activity
The results of the effect of drying on the DPPH radical activity of the elaborated dried products extracted by two extraction techniques: cold soaking and UAE, are presented in Table 1.
According to the results in Table 1 The increase or decrease in AA after drying is mainly attributed to the change in phenolic composition. The same finding was reported by Pradeep and Guha, who reported that the increase in AA after heat treatment is highly correlated with the polyphenolic content. 32 The studies of Dragovic-Uzelac et al., which sought to highlight the correlation between polyphenol content and AA in fruit and vegetable extracts, showed that phenolic compounds contribute directly to the antioxidant capacity. 33 According to Table 1, the results of AA revealed that fresh apricot extracts extracted by UAE method possessed an antiradical activity of 14.37 %, which was much lower than that found by the cold soaking method. The increase in antioxidant activity after drying was mainly attributed to the change in phenolic composition.

Antimicrobial and antifungal activity
The antimicrobial and antifungal activities of fresh, dried apricot, and apricot dough (Prunus armeniaca L.) extracts were estimated by the agar disk diffusion test against four bacterial and two fungal strains. The antimicrobial and antifungal activities of the studied extracts were demonstrated by measuring the diameter of the inhibition zone around the discs.
The inhibition zones of the extracts obtained by the cold soaking method and the ultrasound-assisted extraction obtained by the agar diffusion method show that antibacterial and antifungal activities of fresh and dried apricots depend on the targeted bacteria and fungus. The results are summarised in Figs. 1 and 2.
Most of the methanol extracts from fresh and dried apricots showed biological activity (antibacterial and antifungal activity) against clinical isolates of microorganisms of six species, with zones of inhibition ranging from 4 to 45 mm ( Figs. 1 and 2). The results confirmed that apricots, whether fresh or dried, are potentially rich sources of antimicrobial agents. Yiğit et al. 34 found that apricot kernels have antimicrobial activity against the strains of microorganisms studied (Escherichia coli, Staphylococcus aureus, Proteus mirabilis, Candida glabrata, Candida albicans, and Candida parapisilosis) with zones of inhibition ranging from 8 to 15 mm. In contrast, apricot dough extracts and sample   The lack of antibacterial and antifungal activity of these exceptional extracts towards these strains could be explained by many factors that are related either to the nature of fruit processing or to the wrong choice of techniques, conditions, and tools of manipulation. The poor choice of the adopted method for performing antibacterial activity may be explained by the observation of bacterial resistance. Indeed, Jorgensen and Turnidge found that the well diffusion method was more suitable and reproducible for the evaluation of the antibacterial activity of organic extracts than the agar medium diffusion method. 35 However, the absence of activity of these extracts towards these strains in vitro cannot condition their antibacterial activities in vivo. Some fruit metabolites may be active without being metabolised, and in this case, their in vitro and in vivo activity would be detectable. However, some metabolites are only active after their biochemical reaction; they will be inactive in vitro while they are active in vivo. 36 The results also showed that the activity of the extracts against sensitive bacteria and fungi increased in the extracts of dried apricots treated with sucrose solution, NaCl, and sodium bisulphite. This behaviour can be explain by the effectiveness of our treatment and the extraction methods used.
On the other hand, the fresh apricot extract extracted by UAE showed the highest bacterial activity against Streptococcus sp. at the concentration of 1 g/100 ml with an inhibition diameter of 45 mm. However, apricot dough extract and extracts 9 and 3 showed high activity against Pseudomonas aeruginosa (inhibition zone of 39, 27, and 24 mm respectively, at a concentration of 1 g/100ml), followed by extract 8 against Staphylococcus aureus (inhibition zone of 21 mm, at a concentration of 1 g/100 ml) and finally, the extracts apricot, 6, 5, and 3 against Escherichia coli (zone of inhibition of 17, 17, 16.5, and 16 mm respectively, at a concentration of 1 g/100 ml). Concerning fungal activity, extracts 7 and 8 showed high activity against Candida spp. (inhibition zone of 24 and 19.5 mm respectively, with a concentration of 1 g/100 ml), and finally, extract 8 had activity against Geotrichum capitatum (inhibition zone of 19.5 mm, with a concentration of 1 g/100 ml).
For the cold soaking method, we found that extracts 9 and 4 showed strong bacterial activity against Streptococcus sp. (inhibition zone of 23.5 and 19.5 mm respectively, at a concentration of 1 g/100 ml), followed by extracts 5 and 6 that showed bacterial activity against Escherichia coli (inhibition zone of 16 and 15 mm respectively, with a concentration of 1 g/100 ml), extract 14 showed activity against Staphylococcus aureus (inhibition zone of 14 mm, with a concentration of 1 g/100 ml), and finally, extracts 9 and 2 showed activity against Pseudomonas aeruginosa (inhibition zone of 13 and 12.5 mm respectively, with a concentration of 1 g/100 ml ). Concerning fungal activity, extracts 7 and 8 showed strong activity against Candida spp. (inhibition zone of 24 and 19.5 mm respectively, with a concentration of 1 g/100 ml), and extracts 4 and 6 against Geotrichum capitatum (inhibition zone of 21.5 and 15 mm respectively, with a concentration of 1 g/100 ml).
Streptococcus sp., Pseudomonas aeruginosa, Staphylococcus aureus, Candida spp. and Geotrichum capitatum strains are more sensitive to extracts of samples: fresh apricot, apricot dough, 7, 7 and 8, respectively extracted by UAE with inhibition zones of 48, 39, 25, 24, and 21 mm, respectively. Similarly, it has been reported previously that Candida spp., Streptococcus sp. and Geotrichum capitatum strains are more sensitive to extracts of samples 7, 9, and 4 extracted, respectively, by cold soaking with inhibition zones of 24, 23.5, and 2.5 mm, respectively. By comparing the two extraction methods, we found that the UAE method showed moderate bacterial and fungal activity compared to the cold soaking method. In addition, these results indicate that fresh and dried apricot fruits could not only serve as a rich source of food, but also be of importance in ethno-botanical studies because of their high antioxidant and antimicrobial activities. It can be concluded from this study that the potential use of dried fruit extracts as effective antimicrobial and antifungal agents should be further evaluated. Due to their activity against bacteria and fungi, these extracts could have broad-spectrum antimicrobial properties.

Conclusion
In the present work, Prunus armeniaca L. showed various antioxidant and biological (antibacterial and antifungal) activities desirable for human health. Particularly, the AA of both fresh and processed dried fruits is quite high due to their high polyphenolic content. Hence, the consumption of apricots has been suggested as having health benefits for humans. The results clearly show that the methanolic extracts of fresh and processed dried apricots extracted by UAE and cold-soaking were found to be significantly active against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Streptococcus sp., Candida spp. and Geotrichum capitatum, with different zones of inhibition ranging from 4 to 45 mm, which explains the effectiveness of our treatment and the extraction methods used. These dried fruits exhibited high antioxidant and biological properties, and should thus be recommended for incorporation in foods firstly by increasing their agricultural production leading to the formulation of superior biofunctional foods, and secondly, their consumption should be encouraged. This research is of great importance to advance research on the characterisation and isolation of active molecules contained in this fruit, and thus their exploitation by the pharmaceutical and agri-food industry.
PAPEZA (Laboratory for the Improvement of Agricultural Productions and Protection of Ecosystems in Dry Areas) of the University Batna 1, Algeria.