Antioxidant and anticancer activities of peanut ( Arachis hypogaea L.) skin ultrasound extract

SUMMARY: This study evaluates the effect of ultrasound-assisted extraction on the extractability of polyphenols from peanut skins (PS) and their antioxidant, and anticancer activities. The extraction was performed with solid/solvent ratios of 1:20 and 1:30 (w/v) at ultrasound intensity ranging from 5.8 to 15.4 W/cm 2 for different extraction times (10, 20, 30 and 40 min). The highest polyphenol yield was 167.46 mg GAE/g dried PS. The most abundant polyphenols were catechin, syringic acid, and vanillic acid. The PS ultrasound extract (PSUE) increased the oxidative stability of sunflower oil by four times its initial level. PSUE possessed high inhibitory activity against MCF-7, HepG-2, HCT-116, and PC-3 cancer cell lines, with IC 50 ranging from 1.85 ± 0.13 to 6.1 ± 0.43 μg/ml. In addition, the cytotoxicity of PSUE was examined on HFB4 human normal melanocytes using the MTT assay. These results suggest that PSUE can be used as a natural antioxidant and anticancer agent.


INTRODUCTION
Recent studies indicate that many food wastes are rich sources of bioactive compounds which could be used as nutraceuticals and functional foods (Leichtweis et al., 2021).The peanut (Arachis hypogaea L.) is an important commercial crop used to produce oil.It is an ingredient in peanut butter, confections, and other finished products.The worldwide production of peanuts with shells in 2020 was 53 million tonnes (FAOSTAT, 2022).PS is the pink-red coat which is produced as waste after peanut kernels are roasted and blanched.It represents 3% of the fruit weight.It has limited industrial applications due to its high level of tannins, bitter flavor, low-calorie level, and poor commercial value.However, it is rich in various bioactive compounds which belong to polyphenols.Composite film containing PS polyphenol extract demonstrated DPPH and ABTS radical scavenging activity (Dai et al., 2022).
The oxidation of oils deteriorates the quality characteristics of food during storage.The use of synthetic antioxidants in the food sector is increasingly restricted because they have the potential to cause cancer (Bhadresha et al., 2022).This trend is accompanied by the expansion of the use of natural antioxidants such as polyphenols from plant sources.The cytotoxicity of polyphenols from different plant sources has been investigated in cancer cell lines.Olive pomace methanolic extract showed anticancer activity against HepG2, MCF-7, PC3 and HCT116 cell lines (Mahmoud et al., 2018).Meanwhile, moringa leaf aqueous extract exhibited the same effect on human lung cancer A549 cells (Bhadresha et al., 2022).
Ultrasound-assisted extraction (UAE) is becoming more widely used because of issues with traditional extraction processes.Compared with conventional techniques, the recent ones are more efficient, require less energy, and yield high-quality extracts (Sridhar et al., 2021).Khaopha et al. (2015) indicated that PS methanolic extract obtained by maceration had an anticancer effect against HeLa, HT29, HCT116 and Jurkat cells.
There is a lack of studies about the antioxidant and cytotoxic activities of PSUE which is rich in polyphenols on colon, breast, and prostate carcinoma cells.The objectives of the present study were maximizing the recovery of polyphenols from PS using ultra-sound assisted extraction, the characterization of the phenolic extract, and evaluating its ability to extend the shelf-life of refined sunflower oil, besides its cytotoxicity on different carcinoma cell lines.

Preparation and chemical composition of PS
A PS sample (500 g) was ground and sieved in a 20-30 mesh.The chemical composition of PS powder was determined according to AOAC (2012).The Kjeldahl method was used to determine the total nitrogen content.The protein content on a nitrogen basis was measured using a conversion factor of 6.25.Ether extract was obtained using diethyl ether in a Soxhlet system.The ash content was determined by incineration in a muffle furnace at 550 °C.Crude fiber was determined gravimetrically after digestion of the sample with 1.25% sulfuric acid solution and 1.25% sodium hydroxide solution.

Extraction of polyphenols
Polyphenols were extracted from a roasted peanut skin powder sample with 80% ethanol aqueous solution (v/v).Extraction was performed for 10, 20, 30, and 40 min using a Fisher Sonic Dismembrator (Model 300, USA) and solid/solvent ratios of 1:20 and 1:30 (w/v).Ultrasound power (P) and ultrasound intensity (UI) were calculated using the following equations by Vernes et al. (2019).
Eq. ( 1) Where (dT/ dt) is the increase of °C/min of 200 ml of aqueous ethanol, Cp is the heat capacity of 80% aqueous ethanol (2746 J/kg•°C), and M is the mass (kg) of 80% aqueous ethanol.When the generator was set to 95, 100, 105, and 110 W, the estimated P was 16.5, 26, 37.8, and 43.7 W, respectively.The UI (W/cm 2 ) was calculated as follows Eq. ( 2) where D is the diameter (cm) of the ultrasound probe.Supernatants were saved after extraction and kept at -20 °C until analysis.

Total polyphenols
The total polyphenol content of the PSUE was determined using the Folin-Ciocalteu method as described by Arnous et al. (2002) at 760 nm.Results are expressed as mg gallic acid equivalent (GAE)/g dried PS.

Total flavonoids
The flavonoid content in the extracts was determined as described by Formagio et al. (2014).The absorption of the reaction mixture against the blank was recorded at 510 nm.The analysis was performed in triplicate.Values are expressed as mg quercetin equivalents (QE)/g dried PS.

Identification and quantification of polyphenols using HPLC
The PSUE with the highest level of polyphenols was concentrated at 40 °C under vacuum using a rotary evaporator.The HPLC analysis of the concentrated extract was carried out according to Kim et al. (2006).An Agilent Technologies 1100 series liquid chromatograph equipped with a diode-array detector was used.The Eclipse XDB-C 18 column (150 mm x 4.6 µm x 5 µm) with a C 18 guard column (Phenomenex, Torrance, CA) was also used.Before injection, the sample was filtered through an 0.45 µm Acrodisc syringe filter (Gelman Laboratory, MI).The injection volume was 20 µl.The mobile phase contained (A) acetonitrile and (B) 2% acetic acid.Gradient flow was conducted at 0.8 ml/min.Peaks were ob-served simultaneously at 280, 320, and 360 nm.The peaks were identified by UV spectra and retention times and their values were compared with those of the standards.

DPPH assay
The assay was carried out as described by Brand-Williams et al. (1995).An aliquot (0.2 ml) of the extract was mixed with 2.7 ml of DPPH solution (45 mg/L) and the mixture was kept for 30 min in the dark.The absorbance was read at 515 nm using a Vis-UV spectrophotometer.BHT was used as a reference.The following formula was used to calculate the percentage of radical inhibition: A 0 denotes absorbance in the absence of sample, while A 1 denotes absorbance in the presence of sample.The IC 50 value represented the concentration of the extract required to decrease the initial absorbance of the DPPH solution by 50%.

Reducing power assay
The reducing power of various concentrations of the PSUE was measured according to the method described by Chang et al. (2002) at 700 nm.From the linear regression analysis, the extract concentration which produced 0.5 absorbance (IC 0.5 ) was determined.Results were compared with BHT as a standard.

Quality characteristics of oil
The acid value and peroxide value of the RBD sunflower oil were determined according to the recommended methods of AOCS (2009).The color of the oil samples was measured with the Lovibond Tintometer (Tintometer Ltd., United Kingdom), and a 5.25-inch cell, according to ISO 15305 (1998).

Rancimat analysis
The efficiency of the investigated PSUE (concentrated extract) in protecting sunflower oil against accelerated oxidation was carried out using Rancimat 743 (Metrohm, Switzerland) according to AOCS (2009).The concentrated extract was added direct-ly to the oil samples at the investigated levels.The oil samples enriched with 200 and 400 mg GAE from PSUE (concentrated extract)/kg oil were heated at 110 °C and 120 °C, respectively.Sunflower oil without any added antioxidants was used as a control.BHT was used at a concentration of 200 mg/ kg oil.The oxidative stability was expressed by the induction period (h).The airflow rate was set at 20 L/h.

Determination of anti-cancer activity
The investigated human cancer cell lines and human normal melanocytes (HFB-4) were propagated in DMEM supplemented with 10% heat-inactivated FBS, 1% L-glutamine, and 50 µg/ml gentamycin.All cells were kept at 37 °C in a humidified atmosphere which contained 5% CO 2 .The colorimetric method of Mosmann (1983) was used to assess the PSUE's cell cytotoxicity.The absorbance was read at 570 nm with a microplate reader (SunRise, TE-CAN, Inc., USA).IC 50 values were calculated from a dose-response curve.

Statistical analyses
All analyses were carried out in triplicate, except for the Rancimat analysis (two repetitions) and HPLC analysis (single determination).Statistica software (StatSoft Inc., Tulsa, OK, USA) was used to analyze the variance of the results.The results were presented in terms of means and standard deviation.

Chemical composition of the PS
The ether extract, protein, ash, and crude fiber contents of PS were 8.32 ± 0.22, 11.57 ± 0.27, 2.57 ± 0.08, and 48.04 ± 2.14% on a dry weight basis, respectively.These results supported earlier findings of Muñoz-Arrieta et al. (2021) and proved that PS is a relatively rich source of protein and could be used in the future for the extraction and purification of this nutritional component.PS composition varies with seed maturity and cultivar.

Polyphenol extraction yield
The results in Figure 1a illustrate that increasing extraction time to 20 min at the highest solid/solvent ratio increased the yield of polyphenols, after which the yield decreased significantly (p ˂ 0.05) at each UI used.
The extracted polyphenols with the lowest solid/solvent ratio and at each UI did not significantly (p > 0.05) increase when the extraction duration was extended to 40 min (Figure 1b).The yield in polyphenols increased significantly (p < 0.05) with the increase in UI from 5.8 to 9.2 W/cm 2 during extractions lasting 30 and 40 min, regardless of the solid/solvent ratio.However, increasing UI from 9.2 to 15.4 W/cm 2 did not significantly (p > 0.05) enhance polyphenol extraction.The decrements of extraction yield of polyphenols at higher ultrasonic power could be due to the decomposition of the components (Wang et al., 2018).The maximum polyphenol yield (167.46 ± 0.89 mg GAE/g) was obtained with a relatively high solid/solvent ratio for 20 min at UI of 5.8 W/cm 2 (Figure 1a).
The results of the current study demonstrated that the yield of polyphenols achieved with ultrasonic assistance was superior to that of other researchers (Taha et al., 2012) who relied on conventional techniques (41.5 mg GAE/g dry PS).This proved that ultrasound-assisted extraction is an efficient method for extracting polyphenols as reported by Sridhar et al. (2021).
The results of this investigation indicated that the highest level of flavonoids reached 321.76 ± 2.26 mg of QE/g of PS.Meng et al. (2020) found that the flavonoid content in the PS methanolic extract was 234.33 mg rutin equivalents/g PS.These differences in total polyphenol and flavonoid contents could be attributed to cultivar variations, growing conditions, and extraction techniques.

Identified phenolic compounds in the PSUE
The phenolic composition of PSUE (concentrated extract, 41.55 mg/ml) was analyzed by HPLC.The results are presented in Figure 2 and Table 1.
The PSUE was characterized by a high level of syringic acid and vanillic acid, followed by protocatechuic and p-coumaric acids.Rosmarinic, caffeic, and chlorogenic acids were also identified in the examined extract.The analysis demonstrated that catechin was the most abundant flavonoid compound.This result is in line with the results found by Bodoira et al. (2022).Moderate amounts of kaempferol, chrysin, and apigenin were also detected in the extract.Francisco and Resurreccion (2009) examined the polyphenols composition of skin extracts from three peanut types (Runner, Virginia, and Spanish).They reported that the discrepancy in the polyphenol levels could be due to the differences in peanut cultivar and skin type.

Antioxidant activity
The scavenging activity of PSUE against DPPH radicals is shown in Figure 3a.
The results illustrated the positive correlation between PSUE concentration and its activity against DPPH radicals.Compared to BHT, PSUE had a significantly (p ˂ 0.05) lower scavenging action on DPPH radicals.PSUE inhibited more than 92% of the DPPH radicals at 157.3 μg GAE/ml.However, the IC 50 values of PSUE and BHT were 30.5 ± 0.43 μg GAE/ml and 21.65 ± 0.3 μg/ml, respectively.A decrease in the IC 50 value indicates better antioxidant activity (Brand-Williams et al., 1995).
The reduction in ferric ions (Fe 3+ ) from K 3 Fe (CN) 6 to ferrous ions (Fe 2+ ) by the PSUE is shown in Figure 3b.Increasing extract concentration was accompanied by an increase in reducing power.The results indicated that PSUE had lower reducing power (IC 0.5 =17.51 ± 0.75 mg GAE/ml) than BHT (4.48 ± 0.5 mg/ml).At a concentration of 15.6 μg/ml, PSUE and BHT had reducing power values corresponding to 0.39 ± 0.038 and 0.88 ± 0.057, respectively.These results demonstrate that the PSUE showed concentration-dependent antioxidant activity as reported by Wang et al. (2018).

Oxidative stability of PSUE enriched oil
The acid and peroxide values for the oil were 0.21 ± 0.01 mg KOH/g and 0.73 ± 0.004 meq/Kg, respectively, which supported Codex (2021) requirements for acceptable quality.The results in Table 2 show that the addition of PSUE (concentrated extract) at 200 mg GAE/Kg sunflower oil prolonged the induction period of the oil (tested at 110 °C) by 29%.Meanwhile, enriching oil with BHT at the same level enhanced its stability against oxidation by only 21%.
The addition of 400 mg GAE from PSUE (concentrated extract)/kg oil increased its oxidative stability (measured at 120 °C) by 354%.These results prove that PSUE inhibits lipid oxidation without pro-oxidative effects at a higher concentration.
No significant difference was found in oxidative stability (measured by peroxide value) between sunflower oil samples (control) and those mixed with 0.2% (w/w) of PS ethanol extract, obtained by maceration at room temperature, after 3 days of storage at 60 °C (Larrauri et al., 2016).The addition of 1.56 g GAE of PS subcritical fluid extract/kg chia oil increased its oxidative stability to that obtained with TBHQ at 0.2 mg/kg (Bodoira et al., 2022).
Color is an important oil quality parameter, which increased as the extract level increased.The addition of PSUE at the highest investigated level produced a more highly colored oil (0.8 ± 0.05 Red/4 Yellow) than the oil prepared with half the concentration of the same extract (0.5 ± 0.05 Red/4 Yellow) or 200 mg BHT/kg oil (0.3 ± 0.00 Red/4 Yellow).However, the color of the studied oil samples was less intense than the red and yellow hues of the bleached sunflower oil (2.5 Red/25 Yellow), according to American trading rules as reported by Guliyev et al. (2018).

Cytotoxic activity of PSUE
The cytotoxicity of the concentrated PSUE (concentrated extract, 41.55 mg GAE/ml) was studied on HepG2, HCT-116, MCF-7, and PC-3 carcinoma cells (Figure 4).The inhibitory activity of the investigated extract against HFB4 human normal melanocytes was also assayed.
The PSUE proved more effective than vinblastine sulphate at inhibiting the human cancer cells under investigation.The presence of syringic acid, protocatechuic acid, and catechin might have contributed to the cytotoxic effect of the investigated extract as reported in different studies against cancers of different origins (Elansary et al., 2019;Mihanfar et al., 2021).The values are expressed as mean ± SD of two independent experiments.a The induction time was determined at 110 °C.b The induction time was determined at 120 °C.c Induction time of oil containing antioxidant/Induction time of oil measured at the same temperature.
The investigated extract had a low toxicity (CC 50 = 7.3 ± 0.5 µg/ml) to HFB4 human normal melanocytes (Figure 4e).The selective toxicity levels (IC 50 values for normal fibroblast cells/IC 50 value for cancer cells ratio) for HepG-2 and HCT-116 were 3.9 and 3.80, respectively, showing considerable selective ability (> 2) against those cancer cells according to Valderrama et al. (2016).Rossi et al. (2020) indicated that the PS ethanolic extract did not exert cytotoxicity against human peripheral blood mononuclear cells.
On the other hand, PS methanolic extract was found to have IC 50 values of 10.9 and 19.3 μg/ml on HCT-116 and HepG-2 cancer cells, respectively, but did not affect breast carcinoma cells (Taha et al., 2012).Furthermore, PS methanolic extract which was obtained by maceration induced the apoptosis of HCT-116 cancer cells (IC 50 =50.68μg/ml) (Khaopha et al., 2015).They found that MCF-7 cells were less sensitive to the extract (IC 50 > 90 μg/ml).The results of this study showed that the ultrasound-investigated extract was a more effective anticancer agent than those acquired using other methods from earlier investigations.
Plant-derived medications can advance into clinical trials for further therapeutic development if they are non-toxic to normal cell lines, exhibit cytotoxicity in cancer cell lines, and have high selective toxicity (Rossi et al., 2020).

CONCLUSIONS
At room temperature, a high yield of polyphenols was successfully extracted from PS with aqueous ethanol using ultrasound.Within the examined ranges, increasing UI and duration had a favorable impact on extraction yields.The incorporation of PSUE into sunflower oil at 200 and 400 mg/kg increased its oxidative stability compared to the samples containing the synthetic antioxidant BHT.The extract proved to have in vitro anticancer activity on HepG2, MCF-7, HCT-116, and PC-3 with lower IC 50 values than those of the standard drug (Vinblastine Sulfate).This suggests that PSUE can be safely used in the preparation of functional foods with antioxidant bioactive ingredients.

Figure 1 .Figure 2 .
Figure 1.Total phenolic contents (mg GAE/g dry skin) of the ultrasound extract obtained by, (a) peanut skin/aqueous ethanol ratio of 1:20 (w/v) and (b) peanut skin /aqueous ethanol ratio of 1:30 (w/v).Values are means ± standard deviation of three replicates.Bars with different letters indicate significant difference (p < 0.05) by Tukey's test

Figure 3 .
Figure 3. Antioxidant activity of the peanut skin extract compared to BHT as assessed by: (a) DPPH radicals, (b) Ferric reducing power.The results are represented as average values of three replicates ± SD.Bars with different letters indicate significant differences (p < 0.05) by Tukey's test

Figure 4 .
Figure 4. Cytotoxicity effect of peanut skin extract and Vinblastine sulfate concentrations (µg/ml) against (a) HepG-2 hepatocellular cancer cell line, (b) HCT-116 human colon cancer cell line, (c) MCF-7 human breast cancer cell line, (d) PC-3 prostate cancer cell line, and (e) HFB4 human normal melanocytes.The results are represented as average values of three replicates ± SD.Values with different letters indicate significant differences (p < 0.05) by Tukey's test

Table 1 .
Phenolic and flavonoid compounds of peanut skin ultra- * Not detected.The values refer to a single determination

TABLE 2 .
Induction time and protection factor of sunflower oil enriched with peanut extract at different concentrations