Acetone Extract of Almond Hulls Provides Protection against Oxidative Damage and Membrane Protein Degradation

Abstract

Several studies have revealed that among foods, the consumption of edible nuts has beneficial effects on health which are attributed to their high content of potent antioxidants. Among nuts, the whole seed of the almond (Prunus dulcis) has been demonstrated to possess potent free radical scavenging activity, which is related to the presence of phenolic compounds. The aim of the current study is to evaluate the polyphenol content and the antioxidant ability of almond hull, which is an agriculture solid waste. The present results revealed that among different extraction methods, the acetone extract of almond hulls has a high content of phenolic and flavonoid compounds and a high antioxidant ability, which were determined by using the phosphomolybdenum method and by measuring the potency of the antioxidant, respectively. Moreover, the experimental data disclosed that the acetone extract of almond hulls provides protection against the oxidative damage and the membrane protein degradation that are caused in human erythrocytes by hydrogen peroxide. These phenomena may likely be due to the recruitment of antioxidants by cell membranes and/or translocation to cytosol. Overall, almond hull extract could be considered as a natural source of antioxidants, and its consumption could have a positive effect on human health.

1. Introduction

Phenolic compounds are aromatic secondary metabolites in plants which are found largely in fruits, vegetables, cereals, and beverages, and constitute a main part of the human diet. These compounds are categorized into non-soluble compounds such as condensed tannins, lignins, and cell-wall bound hydroxycinnamic acids and soluble compounds such as phenolic acids, phenylpropanoids, flavonoids, and quinones. All of these groups have received more attention due to their multiple roles in plants and their impact on human health [1].

In fact, there are several pathological conditions which are correlated with perturbation of intracellular redox status such as cancers, cardiovascular diseases, diabetes, osteoporosis, and neurodegenerative diseases. An overproduction of oxidants or free radicals to an extent that overcomes the endogenous antioxidant system brings about a period of oxidative stress which results in a disturbance of signal transductions and consequently biological processes [2,3]. Antioxidants are beneficial compounds that protect cells against oxidative damage by controlling free radical formation. When availability of antioxidants is limited, oxidative damage such as lipid peroxidation, DNA degradation, protein modification, and inflammation become cumulative and threaten human health. Therefore, anti-oxidants that scavenge reactive oxygen species or chelate metal transition ions have great value in preventing the onset and propagation of oxidative diseases [4]. In that respect, epidemiological studies have revealed that long term consumption of diets, rich in plant polyphenols, prevents development of oxidative stress associated diseases by reducing reactive oxygen species production and inhibition of macromolecules oxidation [5,6]. In addition to antioxidant activity of phenolic compounds, several studies have demonstrated that polyphenolic extract also has antimicrobial activity, which has led to it being considered as a good alternative for the conventional antibiotics. Because of these virtues, and also due to their low toxicity and minor microbial resistance to these natural compounds, phenolic compounds are used as natural antimicrobials in the food industry instead of chemical preservatives [7]. Moreover, it has been shown that injection of natural herbs or biologic substances into acupuncture points promotes, maintains, or restores health, and prevents disease. So, phenolic-rich plant extract can also be used in herbal acupuncture [8,9]. Based on these properties, identification and development of such agents has become a major area of experimental studies for diseases.

Almond, scientifically known as Prunus dulcis, belongs to the family Rosaceae, is cultivated in a variety of growing conditions and climates. Although the United States, especially California, is the leading producer of almonds in the world, it is actually native to the Mediterranean climate region of the Middle East, from where then it was dispersed by humans in ancient times to other regions of the world. Almond is an important crop which produces fruits with high commercial value and is currently used worldwide in bakery and confectionery [10,11]. So far, > 30 wild almond species have been recognized which have a bitter taste and eating even a relatively small number of these nuts can be fatal due to the presence of glycoside amygdalin. Indeed, following mechanical damage to the kernel, glycoside amygdalin is transformed into deadly prussic acid (hydrogen cyanide).

It has previously been shown that almond is high in monounsaturated fats, the same type of health-promoting fats which have low-density lipoprotein-lowering effects and reduce risk of heart diseases [12]. Moreover, it has been revealed that almond seed, its brown skin, and green cover (hull) possess radical scavenging potential because of a wide variety of phenolic acids and flavonoids that are predominantly conjugated with sugars or other polyols via O-glycosidic or ester bonds [13,14]. High performance liquid chromatography and gas chromatography-mass spectrometry analyses have been shown that among different parts of almond, the skin and hull are phenolicrich relative to the kernel and shell. Chlorogenic acid and its isomers are the major phenolic compounds in almond hull, however, quercetin, morin, stigmasterol, β-sitosterol, kaempferol, isorhamnetin, and p-coumaric acid were also identified. Quantitative analyses also revealed trace amounts of phenolic compounds in the kernel and shell of almond such as ferulic acid, sinapinic acid, caffeic acid, p-coumaric acid, kaempferol, quercetin, and isorhamnetin [15]. The antioxidant activity of these phenolic compounds in almond, which is mainly due to their redox properties, makes them appropriate candidates in medicine, since oxidative stress eventually leads to many chronic diseases such as arteriosclerosis, cancer, and inflammation [16]. In that respect, according to literature reports, several studies have focused on antioxidant and antiradical characteristics of almond seed and its byproducts in wild or domesticated species from different countries [17–21]. However, there is a lack of information relevant to the antioxidant capacity of Iranian domesticated almond hull in literature. In the present study, the effect of extraction conditions on total phenolic content and free radical scavenging capacity of domesticated almond hull which grows in northeastern Iran was assessed. Moreover, the effect of phenolic-rich extract of almond hull on human erythrocyte membrane integrity was also evaluated.

2. Material and methods

2.1. Materials

Methanol, acetone, ethanol, Folin-Ciocalteu reagent, and sodium carbonate were purchased from Merck (Darmstadt, Germany). Gallic acid, catechin, sodium nitrate, sodium phosphate, and ammonium molybdate were obtained from Sigma Chemical Co. (St Louis, MO, USA).

2.2. Extraction preparation

Almonds were collected from suburbs of Nishabur (Razavi Khorasan province, Iran)in August 2013 and September 2013. The green cover of samples was separated and dried at room temperature away from sunlight. Before the extraction process, almond hulls were ground in a mill. For the extraction with methanol/water (v/v, Me/Wa, 70/30), ethanol/ water (v/v, Et/Wa, 70/30), or acetone/water (v/v, Ac/Wa, 70/30), 2 g of the sample was suspended in 20 mL of the tested solvents for different times and temperatures and filtered through Whatman No. 4 paper (Sigma Chemical Co., St Louis, MO, USA). The solvents were evaporated and the obtained aqueous extracts were dissolved in water to a final concentration of 100 µg/mL and stored in the dark at 4°C for further use. All of the extractions were done in duplicate.

2.3. Determination of phenol content

Total phenolic content was determined by using the Folin-Ciocalteu colorimetric method described previously [22]. Briefly, each sample (0.5 mL) was mixed with 2.5 mL of Folin-Ciocalteu reagent (diluted 10-fold) followed by 2 mL sodium carbonate (7.5%) solution. After incubation at 30°C for 90 minutes, the absorbance was measured at 765 nm by a UV-Visible spectrophotometer (Optizen 322 OUV, Mecasys, Daejeon Korea). The results were expressed as gallic acid equivalents (mg GAE/g dried extract).

2.4. Determination of flavonoid content

The total flavonoid content of each plant extract was estimated by the Nickavar and Esbati [23] method. Based on this method, each sample (250 µL) was mixed with 2 mL of distilled water and 75 µL of NaNO₂ solution (5%). After 5 minutes, 150 µL of AlCl₃ solution (10%) was added and allowed to stand for 6 minutes, and then 500 µL of NaOH solution (1M) was added to the mixture. Immediately, 275 µL of distilled water was added to the mixture. The solution was mixed well and the intensity of pink color was determined at 510 nm. Results were expressed as catechin equivalents (mg catechin/g dried extract).

2.5. Determination of antioxidant capacity by the phosphomolybdenum method

The antioxidant activity of different extracts was evaluated by the phosphomolybdenum method described by Prieto et al [24]. Each sample (0.1 mL, 200 µg/mL) was combined with 1 mL of reagent solution (0.6M sulfuric acid, 28mM sodium phosphate, and 4mM ammonium molybdate). In the case of the blank, 0.1 mL of methanol was used in place of the sample. The tubes were capped and incubated in a water bath at 95°C for 90 minutes. After the samples were cooled to room temperature, the absorbance of each was measured at 695 nm. The antioxidant capacity was expressed as an equivalent of ascorbic acid (mg ascorbic acid/g dried extract).

2.6. Total reducing power determination

The reducing power was quantified by the method of Suseela et al [25]. Each sample was incubated with 2.5 mL phosphate buffer (0.2M, pH 6.6) and 2.5 mL potassium ferricyanide (1% by mass/volume) at 50°C for 20 minutes. Then, 2.5 mL of trichloroacetic acid solution (10% by mass/ volume) was added to terminate the reaction and the mixture was centrifuged at 503 g for 20 minutes. The supernatant was mixed with distilled water and ferric chloride solution (0.1% by mass/volume) and the absorbance was measured at 700 nm. Increased absorbance of the reaction mixture indicated increased reducing power.

2.7. Blood collection and preparation of erythrocyte suspension

Whole blood was obtained from healthy, nonsmoking, and non-drug treatment human adult donors and blood samples were collected in tubes containing anticoagulant ethyl-enediaminetetraacetic acid (EDTA). Red blood cells (RBCs) were isolated by constitutive centrifugation at 503 g for 10 minutes. After removal of plasma and buffy coat, RBCs were washed with saline solution (0.9% NaCl, w/v). Then, phosphate buffered saline solution (PBS, 0.01 M phosphate buffer, pH 7.4, and 0.9% NaCl) was added to the washed RBCs to give a 5% v/v suspension. RBCs were then preincubated at 37°C for 45 minutes with different almond extracts (200 µg/ mL) or ascorbic acid (30 µg/mL, final concentration) and then oxidant (H₂O₂, 10mM) was added to each sample and incubated at 37°C for 1 hour, with gentle shaking. Negative control was prepared by mixing 2 mL of PBS with 2 mL of RBCs suspension, while a positive control was prepared by mixing 1 mL of PBS, 2 mL of erythrocytes suspension, and 1 mL of H₂O₂ solution. Each reaction mixture was used to determine the hemolysis percentage.

2.8. Assay of hemolysis

The reaction mixture was centrifuged at 1,397g for 2 minutes. The absorbance of supernatant was measured at 540 nm using a spectrophotometer. A reference value was determined using the same volume of RBCs in a hypotonic buffer (5 mL phosphate buffer, pH 7.4, 100% hemolysis). The hemolysis percentage was calculated using the following equation:

$${\rm{Hemolysis}}\,{\rm{(\% )}}\,{\rm{ = }}\,{\rm{(A}}\,{\rm{sample/A}}\,{\rm{reference)}}\, \times \,{\rm{100}}{\rm{.}}$$

((1))

2.9. RBCs ghost membrane preparation

RBCs ghost membranes were prepared based on the slightly modified method of Dodge et al [26]. A volume of 50% of RBCs was resuspended in 10 mL of cold PBS (5mM, pH 7.4) and incubated for 30 minutes at 4°C. The suspension was then centrifuged (4,000g,20minutes, 4°C) and the supernatant was discarded. Sedimented cells were then resuspended in cold PBS (1.25mM) containing 2.2mM EDTA, pH 7.4 (ratio 1:15) and incubated for 30 minutes at 4°C. After centrifugation (7,000g, 10 minutes, 4°C), the supernatant was decanted. The pellet (RBCs ghost membranes) was resuspended in PBS containing 2.2mM EDTA and stored at −80°C until analysis. Membrane protein was estimated by the Bradford [27] method.

2.10. Assessment of oxidative damage on erythrocyte ghost membrane proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis

The oxidative modifications on erythrocyte ghost membrane proteins were determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to the slightly modified method of Carini et al [28]. The membrane protein oxidation was induced by addition of 100 µL of 10mM H₂O₂ to 1 mL of reaction mixture containing 100 µg proteins with or without almond extracts (200 µg/ mL) and incubated for 1 hour. Appropriate controls were run along with the test samples. SDS-PAGE was performed on 10% discontinuous gel according to the method of Laemmli [29]. The protein bands were visualized by staining with Coomassie brilliant blue.

2.11. Determination of advanced oxidation protein products levels

Advanced oxidation protein product (AOPP) levels were determined according to the method of Kayali et al [30]. The oxidation of membrane proteins was induced by addition of 100 µL of H₂O₂ (10mM) to 1 mL of each sample with or without almond extracts (200 µg/mL) and incubated for 1 hour. Then, 0.8 mL of PBS was added to 0.4 mL of each sample and incubated for 2 minutes. Potassium iodide (0.1 mL, 1.16M) was added to the sample tube followed by 0.2 mL of acetic acid. The absorbance of the reaction mixture was immediately recorded at 340 nm against the blank solution containing 1.2 mL of PBS, 0.1 mL of potassium iodide (1.16M), and 0.2 mL of acetic acid. The concentration of AOPP for each sample was calculated by using the extinction coefficient of 26lmM⁻¹ cm⁻¹ and the results were expressed as nmol/mg protein.

2.12. Statistical analyses

Data are expressed as mean ± standard deviation of three independent measurements and statistically analyzed using the Student t test. The experimental data were analyzed using one-way analysis of variance and Tukey’s post hoc test, and values of p < 0.05 were considered significant.

3. Results

3.1. Optimization procedure for phenols extraction

Among different methods, solvent extraction is generally used for isolation of polyphenol compounds due to its efficiency and wide applicability. In addition to the selection of appropriate solvent in terms of polarity, other variable parameters such as extraction time, temperature, sample-to-solvent ratio, as well as chemical composition and physical characteristics of the samples significantly impact on the yield of chemical extraction and activity of compounds. Although diversity in structure and composition of the plant materials and the polarity of used solvent impact on the solubility of phenolic compounds, each matrix-solvent system behaves in a way that is not predicted [31–33]. Hence, we designed experimental conditions to evaluate the effects of different solvents methanol/water (Me/Wa), acetone/water (Ac/Wa), and ethanol/water (Et/ Wa), incubation times (2 hours and 6 hours) and temperatures (25°C and 50°C) on polyphenol and flavonoid extraction yield from almond hull. Based on spectrophotometric data presented in Table 1, among six different extraction conditions the highest extraction rate of phenolic compounds from almond hull was obtained (81.0 mg GAEs/g extract) by Ac/Wa solvent, 6 hours incubation, at 50°C and minimum total phenolic content (54.9 mg GAEs/g extract) was obtained by Et/Wa solvent, 2 hours incubation, at 25°C. Moreover, extraction with Ac/Wa solvent, 6 hours incubation, at 50°C led to the highest content of flavonoid (55 mg catechin/g extract), while the low concentration of flavonoid (9.5 mg and 10 mg catechin/g extract) was obtained in Et/Wa solvent, 2 hours and 6 hours incubation, at 25°C, respectively. Although acetone has the least polarity relative to the other solvent it is the best solvent for extraction of phenolic compounds of almond hull. According to the principle of “like dissolves like”, solvent would only extract those compounds which have similar polarity with the solvent [34,35]. It suggests that the most phenolic compounds present in almond hull have moderately polar characteristics. Moreover, the present results revealed that there is a correlation among polyphenol extraction yield, temperature, and incubation time. It seems that 50°C is the best extraction temperature due to enhancing both solubility of solute and diffusion coefficient. Further, mild heating causes softening of plant tissues and disintegration of cell wall. These phenomena favor the release of bound phenolic compounds and augment phenolic extraction yield.

Table 1 Total phenolic and flavonoid content of meth-anolic, acetone, and ethanolic extracts of shell cover of almond in different times and temperatures.

3.2. Antioxidant activity assay

In order to assess the antioxidant activity of almond hull extracts, the phosphomolybdenum method was used. In this method molybdenum (VI) reduces to molybdenum (V) by the antioxidant compounds and green molybdenum (V) complexes form with a maximal absorption at 695 nm [24]. Using this method, the results indicated that the Ac/Wa extract of almond hull (50°C, 2 hours) has the highest antioxidant capacity with a value of 70 mg ascorbic acid equivalent/g dried extract (Fig. 1). The Et/Wa extract of almond hull showed lower activity with a value of 20 mg ascorbic acid equivalents/g dried extract. In contrast to our expectation, the highest antioxidant activity was observed in 2 hours not 6 hours incubation times at 50°C. It has previously been reported that elevation of the extraction temperature would increase the mass transfer of phenolic compounds and also reduce the solvent viscosity and surface tension, and hence promote the extraction of phenolic compounds [28,36,37]. However, prolonged incubation leads to a reduction of antioxidant activity. This may likely be due to thermal destruction of phenolic compounds, reduction in the antioxidant capacities of crude extract, and subsequently disability to scavenge the free radicals [38].

Figure 1

The antioxidant activity of different almond hull extracts. For experimental details see Materials and methods section. Data are presented as mean ± standard deviation, N = 3), p < 0.05. * Significant difference between acetone and methanolic extracts.

3.3. Reducing power assay

In order to measure the reductive ability, Fe⁺³/Fe⁺² transformation in the presence of almond hull extract was assessed [25]. The reducing capacity of extracts may serve as a significant indicator of antioxidant activity. In this assay, antioxidants react by donating an electron, thus converting ferric ions to ferrous ions. Based on the results presented in Fig. 2, the reduction activity of almond hull extracts was lower than the activity of ascorbic acid. However, Ac/Wa extract showed significant reducing potential relative to the other extracts indicating a higher electron donating ability for neutralizing free radicals. Based on these data and evidences presented in Table 1, it seems that high flavonoid content in almond extract is responsible for electron donating to terminate the radical chain reaction.

Figure 2

Reducing power of different almond hull extracts. Data are presented as mean ± standard deviation, N = 3), p < 0.05. * Significant difference between acetone and methanolic extract. ASC = ascorbic acid.

3.4. Protection of RBCs against free radical damage

RBCs are the most common type of blood cell in the vascular system of vertebrates. RBCs are prone to oxidative damage due to the high cell concentration of oxygen and iron (Fe²⁺)-rich hemoglobin, as powerful initiators of the oxidative process [39]. The membrane of RBCs is made up of 39.5% proteins and 35.1% lipids which are highly affected by oxidative stress. In fact, mature red cells have a limited ability to respond to oxidative stress and incurred damages due to incapacity in new protein synthesis and damaged cellular components retrieval. Preservation of membrane structure integrity has a crucial role for efficient function of biological membranes, such as ionic balance between the intracellular and extracellular compartments as well as activity of membrane bound receptors and enzymes. Several studies have recently explored the protective effect of phenolic compounds against RBCs oxidative damage [40,41]. Therefore, the efficacy of almond hull extract in protection of RBCs against hemolysis induced by H₂O₂ was also investigated. Based on the results presented in Fig. 3, hemolysis of RBCs occurs in a time dependent manner when exposing the cells to H₂O₂ (by 45% after 1 hour to 80% after 5 hours). However, preincubation of cells with different almond extracts, significantly, prevents the extent of H₂O₂ induced hemolysis in erythrocyte cells. Among different almond extracts, Ac/Wa extract effectively hinders hemolysis relative to the other extracts. Indeed, acetone extract (50°C, 2 hours) prevented hemolysis by almost 18% after 1 hour to 32% after 5 hours (Fig. 3A) indicating the anti-hemolytic capacity of phenolic and flavonoid components of extract from almond hulls. It has previously been proposed that flavonoids preserve membrane integrity against several chemical and physical stress conditions. Preventing lipid and protein peroxidation is performed by means of a possible interaction between flavonoids and cell membranes [41]. In order to examine this possibility, erythrocytes were treated with Ac/Wa extract for 45 minutes and then cells were centrifuged and washed with PBS. After elimination of phenolic and flavonoid agents which were suspended in supernatant and were not incorporated into cell membranes, the oxidizing agent H₂O₂ was added. As shown in Fig. 3B, there was no significant difference in antihemolytic effect of Ac/Wa extraction of almond hull when suspended polyphenol compounds were removed from the medium as compared with the previous experiment (Fig. 3A). This observation indicated that antioxidant compounds preserve membrane integrity through insertion into cell membranes. In fact, the residence of polyphenol compounds of acetone extract within erythrocyte cell membranes is similar to the action of quercetin on the fluidity, conformational changes of membrane proteins, and the resistance of membrane to hemolytic agents in erythrocyte cells [42].

Figure 3

The percentage of hemolysis in erythrocyte cells induced by H₂O₂ in the presence or absence of different almond hull extracts. (A) The red blood cells (RBCs) were preincubated at 37°C for 45 minutes with different phenolic extracts of almond hull (200 µg/mL) or ascorbic acid (ASC; 30 µg/mL), then the H₂O₂ was added and the percentage of hemolysis was evaluated in a time dependent manner, using a UV/Visible spectrophotometer). (B) The RBCs were treated with acetone extract of almond hull (200 µg/mL) at 37°C for 45 minutes. Cells were washed three times with phosphate buffered saline solution (PBS) and then H₂O₂ was added. After 5 hours incubation, the percentage of hemolysis was evaluated. * Indicates p < 0.05 compared with cells treated solely with H₂O₂. ^† Not significant difference between incubation times.

3.5. Oxidation of membrane proteins

Cellular proteins are one of the prime targets for oxidative stress. Protein oxidation is caused by a variety of reactive oxygen species. Depending on the reactive oxygen species type and content, nitration or oxidation of various amino acid residues occurs which leads to protein-protein cross-linkage and protein fragmentation [43]. In fact, these protein modifications cause modulation of cellular fate towards death.

To gain further insights into the protective effect of almond extract against H₂O₂-induced oxidative damage in RBCs, the extent of oxidative modifications of proteins was estimated by measuring AOPP. As is evident from Fig. 4, there was a significant elevation of AOPP in RBCs (10 ± 0.4 nM/mg protein) when exposed to H₂O₂ for 3 hours. However, preincubation of H₂O₂-treated cells with Ac/Wa extract of almond hull (200 µg/mL) exhibited a significant decrease in AOPP level (by 6.3%). These data are compatible with previous studies in which many natural polyphenol-rich extracts have a protective effect against free radical mediated oxidative damage of proteins [41,44]. Regarding these findings, it could be concluded that there are antioxidant compounds in almond extract which localize within the cell membrane and/or intracellular and act as scavengers of free radicals in the lipophilic environment and prevent proteins from oxidative damage.

Figure 4

Protective effect of different almond hull extracts against protein oxidation induced by H₂O₂. Red blood cells (RBCs) ghost membrane preincubated with different almond extracts (200 µg/mL) at 37°C for 45 minutes. Then, cells were exposed to H₂O₂ for 5 hours. Oxidative modifications of proteins were estimated by measuring advanced oxidation protein products (AOPPs). Data are presented as mean ± standard deviation (N = 3). Data are statistically different at p < 0.05. * Indicates p < 0.05 compared with sample treated only with H₂O₂.

3.6. Evaluation of oxidative damage on erythrocyte ghost membrane proteins by SDS-PAGE

Previous data (Section 3.4 and 3.5) provided substantial evidences that acetone extract of almond hull has protective effects on proteins from oxidative stress. In order to confirm these results, the changes in the pattern of membrane proteins that were produced by oxidative stress were evaluated by SDS-PAGE. Human erythrocyte ghost membrane was prepared and then treated with H₂O₂ in the presence or absence of acetone extract of almond hull. After 1 hour incubation, protein pattern was analyzed on SDS-PAGE. As shown in Fig. 5, protein bands were discernible and distinguishable (Lanes 2 and 3) upon treatment of ghost membrane with H₂O₂ in the presence of almond hull extract compare to control sample (Lane 1). However, treatment of ghost membranes solely with H₂O₂ led to a decrease of protein bands intensity, indicating degradation of ghost membrane proteins. These experimental observations clearly indicate that acetone extract of almond hull contains antioxidant compounds that are responsible for maintenance of erythrocyte membrane integrity and function.

Figure 5

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of electrophoresis of ghost membrane of erythroid cells. Ghost membrane of erythrocytes was pre-treated with acetone extract of almond hull at 37°C for 45 minutes and then H₂O₂ was added. Lane 1: Control; Lane 2: treatment with acetone extract (200 µg/mL); Lane 3: H₂O₂; and Lane 4: H₂O₂ and acetone extract (200 µg/mL).

4. Discussion

In the past decade, the potential health benefits of dietary plant polyphenols as antioxidants have received much more interest. Herein, knowledge has been presented about the phenolic and flavonoid content and biological effects of domesticated almond hull extract which was cultivated in northeastern regions of Iran. The obtained results revealed that the aqueous solution of acetone (70%), extraction temperature, 50°C, and incubation time, 2 hours, were the most efficient for extraction of polyphenols from almond hull. In fact, different operation conditions have been used for extraction of phenolic compounds from the hull of almond which impacted on phenolic content assessment. For instance, Siriwardhana and Shahidi [19] reported 71.1 ± 1.74 mg cate-chin/g extract for total phenolic compounds in the hull part of almond which were extracted with ethanol (80%) at 80°C. In another investigation, the extraction of phenolic compounds from almond hull was performed by methanol (80%) at 80°C resulting in 78.2 ± 3.41 mg GAE/g extract [18]. However, similar to the results in this study, there are several reports in literature which show that aqueous acetone is most effective for extraction of phenolic compounds [45,46].

Based on evidence presented, acetone extract has the most antioxidant and reducing power, which may be due to the high polyphenolic content. In that respect, Takeoka and Dao [47] revealed that almond hull contains polyphenolic compounds such as chlorogenic acid, cryptochlorogenic acid, and neochlorogenic acid which are responsible for high antioxidant activity of hull extract. These data indicate that almond hull is a potential source of these dietary antioxidants [47].

Moreover, it has previously been shown that ion permeability, lipid peroxidation, formation of disulfide bonds, and activation of proteolysis in erythrocyte membrane can be changed by oxidants [48]. In this line, it has been reported that commercially available antioxidant mixture effectively protects erythrocytes from 2,20-azobis-2-amidinopropane dihydrochloride induced membrane protein degradation under the experimental conditions [49]. In addition to pure antioxidant, a mixture of antioxidant compounds in herbal extract is widely used to control erythrocyte damages caused by oxidative stress. Compatible with previous reports, the results presented in this study revealed almond hull extract also has a protective effect against RBCs hemolysis and protein oxidation in erythrocyte membranes due to antioxidant and radical scavenging activity of poly-phenolic compounds of extract (Fig. 6). These conservative concepts of polyphenols may be due to situating of these components within the erythrocyte cell membrane.

Figure 6

Schematic representation of extraction conditions and antioxidant and antihemolytic potential of almond hull. The aqueous solution of acetone (70%), extraction temperature, 50°C, and incubation time, 2 hours, were the most efficient for extraction of polyphenols from almond hull. This extraction condition provides the highest antioxidant and antihemolytic capacity.

Collectively, according to these biochemical evidences, almond green hull can be introduced as a potent source of natural antioxidants which can limit the risk of various oxidative associated diseases. Regarding the high content of polyphenol and antioxidant capacity of domesticated almond hull extract as compare to wild type, it is worthwhile to characterize the bioactive compounds responsible for the observed activities.