Ultrasound Assisted Extraction of Phenolic Compounds from a Jujube By-Product with Valuable Bioactivities

: Jujube plant is a potential source of polyphenols with biological propreties. The purpose of this study was to investigate the application of ultrasound technique for extracting phenolic compounds (TPC) from seeds of Zizyphus lotus under optimization conditions based on response surface methodology. A maximum TPC, total ﬂavonoids content (TFC), and total condensed tannins content (TTC) of 2383.10 ± 0.87 mg GAE / 100g, 486.50 ± 0.38 mg QE / 100g and 15,787.10 ± 0.10 mg CE / 100g, respectively obtained under ethanol concentration 50.16%, sonication temperature 29.01 ◦ C, sonication time 15.94 min and solvent-to-solid ratio 34.10:1 mL / g. The optimized extract was then evaluated for its antioxidant, antiacethylcholinesterase, antihypercholesterolemia, and antiproliferative activities. The results showed that ultrasound method is a green and safe method that can be used to e ﬀ ectively extract TPC from jujube seeds. The biological activity of Zizyphus extract exhibited a very good antioxidant against DPPH (EC 50 = 0.39 µ g / mL) and FRAP (1670.42 ± 6.5 mg / 100 g). Additionally, it possesses acetylcholinesterase (AChE) inhibitory e ﬀ ect (IC 50 = 0.93 ± 0.01 mg / mL) and HMGR inhibition (45.41%) using 100 µ g / mL. The extract signiﬁcantly inhibits cell proliferation on the MCF-7 and HepG 2 tumor cell lines with an IC 50 values of < 0.05 and 3 ± 0.55 mg / mL, respectively. Therefore, the ultrasound method can be considered a method for obtaining a signiﬁcant anticancer activity with respect to the lines and therefore makes it possible to recover a maximum of phenolic compounds in less time with an AChE and HMGR inhibitory activity. Thus, it can be suggested that Zls extract is a promising fruit for the development of supplementary dietary due to its potential behaviour as nutraceutical. sonication temperature 29.01 ◦ C, sonication time 15.94 min, and solvent-to-solid ratio 34.1 / 1 mL / g, giving a maximum TPC yield of 2406.0835 mg GAE / 100g. The Zls extracts exhibited a signiﬁcant antioxidant e ﬀ ect with both DPPH and FRAP assays, with a high inhibitory e ﬀ ect for AChE and HMGR tests and antiproliferative capacities against MCF-7 than HepG 2 cell lines. However, this study proved that application of ultrasound technology was e ﬃ cient for obtaining maximum yield of bioactive compounds from jujube seeds in a shorter time when compared to traditional method which can be used by exploiting the antioxidant properties (food additives, nutraceuticals, etc.), antiacetylcholinesterase, antihypercholesterolemia, and antiproliferative properties which may be useful in the development of new strategies to treat Alzheimer, hypercholesterolemia diseases, and cancer. Further studies are needed to identify active compounds after puriﬁcation of extract and evaluate the cytotoxic mechanism of Zls extracts with in vivo models which be enable to production on an industrial scale (food, pharmaceuticals, and cosmetics).


Introduction
The production of free radicals (FR) is associated with many physiological and biochemical processes that have taken place in the human body and their over production leads to the appearance of and nutraceutical applications. To date, no data have evaluated the effect of ultrasound on polyphenols extracted from Zizyphus lotus seeds (Zls) and evaluation of its antioxidant, antiacetylcholinesterase, antihypercholesterolemia, and antiproliferative properties. The overall aim of this research work was to find out optimal extraction parameters (ethanol concentration, time, temperature, and solid/liquid ratio) for the valorization of phenolic compounds extracted from jujube seeds using green extraction technology under RSM model through the investigation of some biological effects of UAE extracts. This could be one of the economical solutions to valorize vegetable biomass in order to protect consumer and environment by consuming natural products and avoiding the generation of some industrial wastes. Additionally, it is one of the promotional strategies to meet the challenge in the 21st century through its uses on the development of new functional and nutraceutical ingredients in food and pharmaceutical industries with a high nutritional value.

Plant Materials and Chemicals
Jujube samples (Zizyphus lotus L.) were collected from Djelfa province of Algeria. Jujube seeds were cleaned and separated manually. Seeds were dried at 40 • C for 24 h in an oven (Memmert, Modell 100-800, Schwabach, Germany). The dried seeds were milled into fine powder (<250 µm) which were frozen stored at 4 • C until analyses.

Ultrasound Assisted Extraction
One gram of powder material was dissolved with the extracting solvent, introduced in an amber glass vial, and placed in a ultrasonic bath (J P.SELECTA 195W 50/60HZ, Barcelona, Spain) at 20 kHz. As previously stated, the suspension was exposed to ultrasonic waves allowing the appearance of cavitation pellets (Table 1). This last being very sensitive, was controlled by external frozen water circulation. After extraction, the extract was filtered through Whatman No. 1 paper and kept in shaded flasks at 4 • C for further analysis.

Response Surface Methodology (RSM)
In current study, a Box-Behnken design (BBD) experimental design with a total of 27 experiments based on the response surface methodology (RSM) was used to evaluate the effect of the independent variables including X 1 -Solvent concentration (% v/v), X 2 -Sonication temperature ( • C), X 3 -Sonication time (min), and X 4 -Solvent-solid ratio (mL/g). The concentrations are indicated in Table 1 and used for each test as indicated in Table 2, Total Phenolic Content (TPC) was determined and used for estimating the parameters in the following equation. After estimating these parameters, the prediction of the of the TPC yields on jujube seeds extracts was performed following the second order experimental model according to Equation (1).
where y represents the predicted response which is TPC yield; βo is a constant coefficient; βi, βii, βij are regression coefficients for linear, quadratic, and interactive terms, respectively; and Xi and Xj represent the coded independent variables while E is error. The significance and suitability of the model was analyzed by the variance (ANOVA) using JMP.

Total Phenolic Content (TPC)
The TPC yield was determined for Zls extracts according to the color method based on Folin-Ciocalteu reaction [19]. The results were expressed as mg gallic acid equivalent per 100 g of dry matter (mg GAE/100 g).

Total Flavonoids Content (TFC)
The TFC yield was measured for Zls extracts, using methanolic aluminium chloride (AlCl 3 ) for 2% as described in Ghafar, et al. [33]. The results were expressed as mg quercetin equivalents per 100 g of dry matter (mg QE/100 g).

Total Condensed Tannins Content (TTC)
The TTC yield was determined using the method of Hagerman [34] and the results were expressed as mg catechin equivalent per 100 g of dry matter (mg CE/100 g).

Antioxidant Activity by DPPH and FRAP Assays
The radical-scavenging activity (DPPH) and ferric reducing antioxidant power (FRAP) of Zls extracts was measured by the methods adapted from those reported by Hammi, et al. [35]. Results were expressed as the percentage of DPPH, calculated following the equation below (Equation (2)): where A b, A s , and A DPPH were the absorbance value of blank, diluted extract, and control, respectively. The radical-scavenging activity (EC50) is defined as the concentration of Zls extracts that provided 50% of DPPH free radicals. However, FRAP assay was expressed in terms of antioxidants having an iron reduction capacity equivalent to that of equivalents in gallic acid of 1 mg per 100 g of dry matter (mg GAE/100 g).

AChE and HMGR Inhibitions Tests
Anticholinesterase activity was determined using AChE assay according to what was previously reported [36]. Briefly, 100 µL of distillated water was added to 325 µL of 50 mM Tris buffer (pH 8) and 25 µL of AChE solution. The solution was mixed and incubated for 15 min at 25 • C. Subsequently, 75 µL of AChI solution (mg/mL) and 475 µL of DTNB (1.2 mg/mL of 50 mM Tris buffer (0.1 M NaCl and 0.02 M MgCl 2 ) at pH 8) were added. The absorbance was read for 4 min with 30 s intervals and the initial velocity was calculated at 405 nm on the spectrophotometer Schimadzu UV-160A, Kyoto, Japan. A control reaction was carried out using the same procedure but with adding 100 µL of distillated water instead of 100 µL of Zls extract at different concentrations. All tests were done in triplicate and the percentage inhibition was calculated as where I is the percent inhibition of acetylcholinesterase, Vsamples the initial velocity of the extract containing reaction, and Vcontrols the initial velocity of the control reaction. The inhibition of the enzymatic activity of 3-hydroxy-3-methylglutaryl reductase (HMGR) was measured using the oxidation of nicotinamide adenine dinucleotide phosphate hydrate (NADPH) in triplicate [37]. Briefly, at 0, 1, 2, 4, and 6 min, aliquots were removed and then the reaction was stopped by adding methanol (50%). The amount of NADPH wasanalyzed by HPLC-DAD using VWR-Hitachi Elite LaChrom ® , Tokyo, Japan. In order to evaluate the decrease of the peak area over time, which allowed to make a linear regression and to obtain the reaction velocity, the decrease in activity was monitored at 340 nm. The activity value without inhibitor was considered to be 100%. The IC 50 values were determined from regression curves by plotting inhibition as a function of the concentration of inhibitor.

Anti-Proliferative Activities on HepG 2 and MCF-7 Cells
The cytotoxicity of Zls extracts were performed using the MTT viability test, against the human tumor cell lines HepG 2 (ATCC#HB-8065), from human hepatocellular liver carcinoma cell lines, and MCF-7 HTB-22), from breast cancer, were cultured in DMEM supplemented with 10% FBS, 100 U/mL Pen-Strep, and 2 mM L-glutamine at 37 • C in an atmosphere containing 5% CO 2 every 48 to 72 h before reaching confluence, the medium was changed [10]. For each concentration of extract, the assays were done in 8 × 12 replicates and the cell viability percentage was calculated by the following equation (Equation (4)

Statistical Analysis
In order to see the effect of UAE obtained from the Box Behnken Design (BBD) trials, ANOVA analysis was used in this study. Each extraction trial and all the analyses were carried out in triplicate. The JMP (Version 10.0, SAS) was used to construct the BBD and to analyze ANOVA results.

Model Parameter Estimation
The factorial experimental design based on a BBD using UAE method and corresponding responses for the obtaining of TPC from Zls extracts was investigated and presented at various conditions with a model of the total of 27 experiments in Table 2. In order to estimate experimental error measurement, three replications at the central points were made [38]. The factors studied were ethanol concentration, sonication temperature, sonication time, and solvent/solid ratio, which is ethanol/seed quantity. These factors were used in the in the range indicated in Table 1. The parameters were estimated from the experimental TPC results and ANOVA analysis was carried out to determine the applicability of the model, Table 2.
The second order polynomial equation was generated to describe the empirical relationship between the Zls extract and operational conditions in terms of coded values. The mathematical models were simplified by neglecting statistically the insignificant terms (p > 0.01) following predictive equation: It can be seen that all factors influence the extraction yield as linear and quadratic effects, but the interaction effects were significant for all the four parameters, positive for ethanol concentration and sonication temperature , negative for ethanol concentration and sonication time. While, positive also for ethanol concentration and solvent-solid ratio which was highly significant then the others. On the other side, it can be seen in Equation (3) that X 2 X 3 , X 2 X 4 , X 3 X 4 didn't exhibit any significant effect on extraction yield (Table 2).
However, if there is any significance of each factor, it was demonstrated by a presence of p < 0.05 and the contrary, it was also demonstrated p-values (p < 0.001) which indicates high significance. Very low p-values (p < 0.0001) indicated that each generated model was statistically significant and suggests that the UAE of Zls could be well described with those appropriate models.
The values of R-squared are close to 1 for the model (0.98 and 0.95 for R 2 and R 2 , adjusted respectively), which are very high and indicates a good correlation between the experimental and the predicted values, also indicated that 98% could be explain by the model of the variation in the TPC extracts using UAE method. In addition, other parameters were insignificants as F-value for the lack of fit (p > 0.05) and values of coefficient of variation (CV = 4.11) which provide also the validity of the deduced model.

Effect of Experimental Conditions on TPC, TFC, and TTC Extraction Yield
The influence of the four parameters on TPC yields is shown in Figure 1. Confirming the results of the single-factor trials, Figure 1a-c shows that the TPC yield reached a maximum level when ethanol concentration was set at medium levels (0 coded value) as it mentioned in Table 2, we can notice that the yield of TPC using UAE mainly depends on the ethanol concentration as its quadratic, interaction and linear effects were highly significant (p < 0.01), which showed the increase on TPC for all other parameters.
Figure 1c-f shows that the interaction effect of temperature with other factors on TPC yield that was very limited and stable as demonstrated in the equation model. Thus, only the Figure 1c showed a positive influence of both ethanol and temperature on the TPC extraction and the decrease of TPC recovery from the other figures is mainly associated to their thermal degradation at higher temperature.
However, Figure 1b-e shows that maximum extraction TPC yield was for 2100 mgGAE/100 g when using a ratio about 30 (mL/g, v/w) over a range of temperature and time factors, in contrary in interaction with ethanol concentration which was very significant as confirmed also as positive effect in Table 2. The increase in TPC was deemed by the effects of acoustic cavitation that contribute to the formation and rupture of cavitation bubbles and then facilitate the mass transfer of the process, while, TPC started to decrease after higher increase in ratio which may affect the dispersion of the ethanol under ultrasound energy density [39].
Furthermore, Figure 1f shows the interaction effect between temperature and time sonication. It is obvious that longer sonication time increases the extraction yield for polar compounds in hydro-alcoholic solvent. While, the prolonged exposures at higher temperature decrease significatively the TPC recovery due to their thermal degradation which confirmed the results of Table 2. The prediction values for the optimal TPC extraction were verified experimentally. Optimal conditions resulted in ethanol concentration 50.16%, sonication temperature 29.01 • C, sonication time 15.94 min and solvent-to-solid ratio 34.1/1 mL/g with a predicted TPC yield of 2406.08 ± 79.87 mgGAE/100 g (Figure 2). TPC started to decrease after higher increase in ratio which may affect the dispersion of the ethanol under ultrasound energy density [39].
Furthermore, Figure 1f shows the interaction effect between temperature and time sonication. It is obvious that longer sonication time increases the extraction yield for polar compounds in hydroalcoholic solvent. While, the prolonged exposures at higher temperature decrease significatively the TPC recovery due to their thermal degradation which confirmed the results of Table 2.
The prediction values for the optimal TPC extraction were verified experimentally. Optimal conditions resulted in ethanol concentration 50.16%, sonication temperature 29.01 °C, sonication time 15.94 min and solvent-to-solid ratio 34.1/1 mL/g with a predicted TPC yield of 2406.08 ± 79.87 mgGAE/100 g (Figure 2). The UAE was carried out at these optimal conditions obtaining a TPC yield of 2383.10 ± 0.87 mgGAE/100g, very close to the value predicted by the model (Table 3). This result indicates that Zls showed a significantly higher TPC recovery in comparison to TPC obtained from other plants. In the meanwhile, the ethanol/water mixture showed a high extraction efficiency by ultrasound and this was in agreement with other studies showing a great effect of ethanol concentration for phenolic compounds extraction, from Citrus limon fruit using 63.93% of ethanol (1502.2 ± 0.88 mgGAE/100 g) The UAE was carried out at these optimal conditions obtaining a TPC yield of 2383.10 ± 0.87 mgGAE/100g, very close to the value predicted by the model (Table 3). This result indicates that Zls showed a significantly higher TPC recovery in comparison to TPC obtained from other plants. In the meanwhile, the ethanol/water mixture showed a high extraction efficiency by ultrasound and this was in agreement with other studies showing a great effect of ethanol concentration for phenolic compounds extraction, from Citrus limon fruit using 63.93% of ethanol (1502.2 ± 0.88 mgGAE/100 g) [32]. Similarly, from Pistacia lentiscus leaves that obtaining a lower TPC value of 1420.76 ± 19.98 mgGAE/100 g [40]. More recently, Esmaeelian, et al. [41] have shown a lower TPC value from Crocus sativus L. corms extract than our result (100.39 mgGAE/100 g dry saffron corm) using 80% of ethanol. The extraction process used in the present work give higher TPC than Indian jujube cultivars using conventional extraction method ranging from 172.08 to 328.65 mgGAE/100 g with 80% of ethanol [42]. Similarly, higher TPC recovery than work of Al-Saeedi, et al. [43] from Oman Zizyphus jujuba fruit which has a content of 64.89 ± 0.44 mg GAE/100 g using methanol as extraction solvent. More recently, Noriega-Rodríguez, et al. [44] found a TPC value of 2.16 g gallic acid equivalent (GAE)/100 g from Globe artichoke (Cynara scolymus L.) using conventional extraction method with 75% of ethanol which is lower than jujube extract. To note, these results show the efficiency of extraction of the phenolic compounds using the hydro-alkolic solvent which makes it possible to increase the polarity, and thus the solubility of the solid ratio that increased the extraction yield beside the other factors.
Regarding to the TTC yields, the Zls extract was for 15,787.10 ± 0.10 mg CE/100 g (Table 3), which is five times higher than that of polymeric proanthocyanidins extracted from Zizyphus jujuba fruits, which was between 939 and 2548 mg/100 g in depends on cultivars [47]. The present work suggests that UAE method is an efficient alternative to other extraction techniques for extracting and maximizing polyphenols from Zls in short extraction time and jujube seeds is a non-negligible source of polyphenols.

Antioxidants Activity of Zls Extract
The imbalance between the production of reactive oxygen species (ROS) and the biological system's antioxidant defenses is defined as the oxidative stress. This proved in several studies to develop a lot of diseases. In order to protect our human body from these diseases, the antioxidants are showed to be effective for neutralization of free radicals [14]. However, to determine whether the UAE extraction process impact the biological functions, the antioxidant effects of jujube seeds extracts were examined and evaluated by DPPH radical scavenger and FRAP model ( Table 3).
The DPPH scavenging assay of the Zls extract revealed a significant highest activity with lowest EC 50 value (0.39 µg/mL) than that of Tunisian Z. lotus leaves and fruits extract using methanol (0.10 ± 0.001 and 0.31 ± 0.005 mg/mL) [48], Tunisian Z. lotus pulp and peel extracts using UAE with an EC 50 of 0.28 mg/mL [19], from Z. mucronata roots (0.029 ± 0.05 mg/mL) [49], and from Korian Z. jujuba seeds (mechu and sanzoin) (0.3 and 0.1 mg/mL), respectively [50]. Overall, this study showed that Zls extracts exhibited a high antioxidant effect in comparison to some Zizyphus species using both UAE and conventional methods. Furthermore, Zls extract by UAE exhibited a significant iron-reducing power (1670.42 ± 6.5 mg/100 g) and was found to be higher than those obtained with 471.6 ± 30.8 mg/100 g from Z. jujuba cv. Zaowangzao [51].
Thus, this paper revealed the effectiveness of ultrasound method for extraction of polyphenols from jujube seeds with significant antioxidant activities in comparison to conventional methods that gave lower recovery and activity which could be attributed to the mechanic cavitation of ultrasound due to the acoustic bubbles which results to enhanced desired compounds without altering its quality. The inhibition of DPPH as well as the iron chelating effect are probably due to the significant jujube content in photochemical substances especially in phenolic compounds, the latter having powerful reducing effects of oxidation. In addition, from other jujube extracts that contain other compounds namely ascorbic acid, tocopherol, and pigments, were found to present synergic effects between them and contribute to the total antioxidant activity of this extract, and therefore the trapping of free radicals. While, this suggested that not only phenolic compounds present in the jujube extract can act as antioxidants but also other compounds may be responsible for this activity [52][53][54]. In our case, the antioxidant activity using DPPH and FRAP methods from Zls extract is in perfect agreement with other research showing that there can be correlation between the phenolic content and the antioxidant capacity [55].

AChE Inhibition of Zls Extract
The antiacetylcholinesterase activity of the Zls extract are presented in the Table 3. The rapid hydrolysis of ACh following the stopping of the transmission of nerve impulses to cholinergic synapses is well controlled by the role of AChE. One of the ways deemed effective used against AD is more particularly based on the inhibition of AChE, which makes it possible to maintain the levels of acetylcholine for the transmission of nerve impulses [56]. The anticholinesterase activity of jujube extract at different concentrations exhibited inhibitory activity with an IC 50 value of 0.93 ± 0.01 mg/mL. These results are higher than that obtained from Zizyphus oxyphylla extracts using nbutanol which showed a maximum inhibitory effect with an IC 50 value of 9.58 ± 0.08 mg/mL [57]. This demonstrated the good effect of the used method mainly the good extraction yield of many compounds from Zls, ethanol/water used for extraction and ultrasounds cavitation of Zls which exhibited a good inhibition of AChE which can be applicable against AD. Ethanol concentration is considered as a crucial factor in UAE due to cavitation phenomena enhancing solvent penetration into jujube extract [32]. Moreover, the Zls extract is approximatively in the same range than Tunisian Zls extract [58] using acetone for extraction by maceration method (0.85 mg/mL). However, in comparison to other plants that used acetone also, our extract had a highest AChE inhibitory effect than that obtained from Herniaria fontanesii and Hyoschy amusalbus with an IC 50 value of 1 and 1.17 mg/mL, respectively [58]. Thus, our results suggested that extraction using ultrasounds under different conditions mainly ethanol as solvent extraction is an important parameter to take into account in AChE tests. Furthermore, Fucus vesiculosus extracts from Tagus estuary presented an IC 50 of 840.85 µg/mL which is in well agreement than our AChE inhibitory from Zls. In addition, the standard galantamine showed an IC 50 of 0.14 µg/mL that is lower than these extracts [59]. These suggests that the intestinal motility may be affected by its consumption although with a much less effect comparatively to the chemical drugs [37]. Major medicinal plant extracts showed some level of inhibitory activity against the AChE. This could be attributed to the phytochemicals mostly phenolic compounds present in the extract and their possible synergistic interaction effect [59][60][61][62][63][64]. To the best of authors knowledge, this is the first report on in vitro inhibition of the AChE enzyme by Zls extracts under the effect of ultrasound extraction where the interest of its application against Alzheimer's disease.

HMGR Inhibition of Zls Extract
The HMGR inhibition by the Zls extract is demonstrated, at the concentration of 100 µg/mL Zls ethanolic extracts showed an activity of 45.41% as a HMGR inhibitors which is higher than the acetone and ethanol extracts from lichen U. complanata with 2.22 and 21.48%, respectively, at the concentration of 60 µg/mL [12]. Similarly, Peganumarma and Tencriumpolium from aerial parts extracts showed a value of 28.5 and 28.8% of HMGR inhibition which are lower than our sample [65]. However, Zls extract showed a significant HMGR inhibitory effects than seeds extracts of some species, mainly Cannabis sativa (7.4%), Cuminum cyminum (26%), and Pimpinella anisum (10.5%) [65]. The IC 50 of the drug simvastatin was found to be around 0.198 ± 0.015 g/mL, which is lower than the value determined for the Zls extract, therefore a higher activity than that observed in our extract. These are in good agreement to our previous results from Centaurium erythraea extracts [37]. However, this drug is a pure compound while Zls is a mixture of several compounds some of which may show significant activities but which are masked in the mixture [59]. Several bioactive compounds mainly polyphenols, saponins, alkaloids, and triterpenes were found to have a good hypolipidemic activity against HMGR [66][67][68]. Thus, jujube extract was found as a modest HMGR inhibitors which is due to the presence of some bioactive compounds that may be responsible for this enzyme inhibition like all polar plant extracts found in literature [66,67]. The properties of our sample phytochemicals make them possible antihyperlipidemic applications by fitting the enzyme active site.
Furthermore, other Zizyphus species (Z. mauritiana) leaves extracts were used previously for the treatment of fatty liver and atherosclerosis by reducing cholesterol and triglyceride and levels [69]. Among some studies that have shown the effect of polyphenols in inhibiting the action of HMG-CoA, demonstrated that curcumin, tetrahydrocurcumin, epigallocatechin-3-gallate, and kaempferol among all the other polyphenols tested can occupy the HMG-CoA binding site on the NADP+ site which utilizes two molecules of nicotinamide adenine dinucleotide phosphate-oxidase (NADP[H]); thus, can play the role of competitive inhibitors of substrate binding to enzyme that can block the electron transfer on the substrate HMG-CoA [70]. Compelling effect of these compounds and major phenolic compounds in general indicates the importance of their uses for the cholesterol-lowering in order to the maintenance of cardiovascular health [71,72].

Anti-Proliferative Activity of Zls Extract
The in vitro evaluation of Zls extracts on cytotoxicity effects were analyzed. The toxicity of extracts was tested in the human cell lines HepG 2 and MCF-7 using 5 serial concentrations ranging from 0.05 to 1 mg/mL of extract in order to calculate the cell viability. The IC 50 values which confirm the concentration of extracts that killed 50% of the cells was obtained from dose-response curves. The phenolic compounds of Zls studied were revealed to be non-toxic towards only to HepG 2 cell line because the value is higher than 0.1 mg/mL. This value is considered as limit of toxicity to human cell lines [73]. In contrary, Zls extract analyzed against toxicity in MCF-7 cells showed an IC 50 value lower than 0.05 mg/mL which is considered toxic to human cell lines. The value of 1 mg/mL which correspond to the maximum concentration present in the UAE extract inhibited 70.84% and 26.21% for MCF-7 and HepG 2 cells, respectively. Thus, Zls extract exhibited no significant activity against HepG 2 cells. In contrary, it exhibited a significant activity against MCF-7 cells. These findings can be due to the main bioactive compounds contained in Zls extract that may include flavonoids, tannins, alkaloids, terpenoids, and saponins as observed previously from other jujube species and from other part of Zizyphus lotus which showed a strong antiproliferative activity against HepG 2 and MCF-7 cells [21,74].
In addition, Z. jujube fruits extract was found to exhibit activity against MCF-7 cells with an IC 50 value of 1.8 mg/mL after 24 h which is higher value than that of Zls extract (IC 50 less than 0.05 mg/mL after 24 h) [75]. The results of cytotoxic action of Z. jujuba extract indicated a reduction inviability and high potent inhibitory effect toward the proliferation of MCF-7 cells, this effect may be due to cell apoptose [76]. However, the cytotoxicity effect reported from Zls extracts against HepG 2 cells was significantly higher than that reported from mung bean sprouts extracts obtained using maceration method with an IC 50 of 14.04 ± 1.5 mg/mL. This extract also contained polyphenolic compounds [77]. The effective cytotoxicity toward hepatocellular HepG 2 and beast MCF-7 could be related to the presence of the some secondary metabolites, some of them are considered the major class of jujube polyphenols, represented from 89 to 94% of the total phenolic contents mainly flavan-3-ols such as monomer (as (-)-epicatechin, gallocatechin gallate, and (+)-catechin), dimer (procyanidine B2), and polymeric proanthocyanidins in Zls, which were applicated in the inhibition of cell proliferation in different cancer types [47]. Quercetin-3-O-rutinoside found in other parts of Z. lotus, which represents 50% of jujube flavonoids offers a plausible explanation of the observed cytotoxicity [74].
The present research data suggests that in some cases the ultrasound extraction can positively influence the extraction yield of TPC, TFC, and TTC, also the antitumor activity against the tested cell lines. This is can be related to the mechanical acoustic effects of ultrasound which causes the rupture of the cell wall allowing mass transfer, and therefore increase the recovery process of TPC but following the sonication time (15.94 min), which is a very sensitive parameter in extraction procedure. To the best of our knowledge, no study has as yet been carried out on the effects of ultrasounds on phenolic extracted from Zls and cytotoxicity toward hepatocellular HepG 2 and beast MCF-7 of the studied UAE extract from Zls.

Conclusions
In this study, the effect of ultrasound on phenolic compounds extracted from jujube seeds based on the BBD model was employed to improve the extraction yield and bioactivities of Zizyphus lotus seeds extract. The optimal conditions of UAE were determined as ethanol concentration 50.16%, sonication temperature 29.01 • C, sonication time 15.94 min, and solvent-to-solid ratio 34.1/1 mL/g, giving a maximum TPC yield of 2406.0835 mg GAE/100g. The Zls extracts exhibited a significant antioxidant effect with both DPPH and FRAP assays, with a high inhibitory effect for AChE and HMGR tests and antiproliferative capacities against MCF-7 than HepG 2 cell lines. However, this study proved that application of ultrasound technology was efficient for obtaining maximum yield of bioactive compounds from jujube seeds in a shorter time when compared to traditional method which can be used by exploiting the antioxidant properties (food additives, nutraceuticals, etc.), antiacetylcholinesterase, antihypercholesterolemia, and antiproliferative properties which may be useful in the development of new strategies to treat Alzheimer, hypercholesterolemia diseases, and cancer. Further studies are needed to identify active compounds after purification of extract and evaluate the cytotoxic mechanism of Zls extracts with in vivo models which be enable to production on an industrial scale (food, pharmaceuticals, and cosmetics).