Ellagic Acid Displays a Protective Effect on Red Blood Cell Membrane During Aging in Humans

Abstract

Ellagic acid (EA) is an antioxidant and anti-inflammatory compound primarily found in pomegranates and berries. It is a dimer of gallic acid and has been shown to have health advantages. We assessed the protective effect of EA against oxidative stress caused by tert-butyl hydroperoxide (t-BHP) on erythrocyte cell membrane indicators during the aging process in humans. In control and oxidative stress caused by t-BHP in human subjects' erythrocytes of both sexes (n = 80), we evaluated the in vitro effect of EA on Na⁺, H⁺ exchanger (NHE), Na⁺, K⁺ ATPase, Ca²⁺ ATPase, protein carbonyl group content and osmotic fragility. We note that the NHE activity increases relative to their respective controls in the young, middle, and old subjects. Stress-inducing t-BHP significantly increases (p < 0.001) NHE activity in all age groups compared to the respective control. NHE activity is further reduced by EA treatment. In middle-aged and older human participants, erythrocyte membrane Na⁺, K⁺, and Ca²⁺ ATPase activity was considerably (p < 0.001) lower than in younger subjects in the control group. In all age groups, there was a noteworthy reduction (p < 0.001) in Na⁺, K⁺, and Ca²⁺ ATPase activity in the membrane of t-BHP-induced erythrocytes compared to their corresponding controls. Administration of different concentrations of EA (10⁻⁵–10⁻⁷ M) decreases carbonyl group content significantly compared to their respective control in each age group in a concentration-dependent manner. When the erythrocytes were induced with stress using t-BHP, the lysis % increased, and after the treatment of EA, the lysis % was found to decrease in all the age groups. The activity of membrane-bound enzymes is markedly increased when EA is applied to these erythrocyte membranes. Through the modulation of membrane transporters, the study sheds light on the beneficial health advantages of EA.

Introduction

Over a lifetime, aging is a complex process that alters how an organism function. Aging is brought on by molecular and structural changes that occur in cells, tissues, and organs [1]. An aging population results from both population growth and improved medical facilities. The elderly populace is predicted to treble and exceed two billion between 2015 and 2050. One in five individuals will be 60 or older by 2050 [2]. Critical cellular functions are harmed, the rate of mutations rises, and oxidative stress becomes lethal [3]. Although more than 300 theories have been proposed to explain what causes aging, they have yet to be conclusively shown. The free radical theory of aging, by Prof. Denham Harman (presented in 1956), has influenced and given directions to aging research for almost five decades [4]. A variety of endogenous and external activities produces reactive nitrogen and oxygen species. The cellular system's antioxidant defence mechanisms neutralise these reactive substances. A disparity between antioxidants and oxygen species defence systems causes oxidative stress. Age-related illnesses arise from the functional decrease of tissues and organs caused by oxidative damage [5].

A class of phytochemicals with phenolic rings known as polyphenols are primarily found in a wide range of plant-based goods. When consumed in the diet, these polyphenols treat many age-related illnesses [6]. Polyphenols, bioactive compounds found in nature, have been researched as powerful anti-aging agents. These substances control inflammation, oxidative stress, cell senescence, and autophagy-induced damage. Most polyphenols have drawn interest because they guard against age-related illnesses like cancer, neurological disorders, and cardiovascular ailments [7].

EA is a phenolic molecule found in nature that is a dimer of gallic acid and naturally occurs as a secondary metabolite in plants [8]. The compound's chemical formula is C₁₄H₆O₈, and its molecular weight is 302.19 g/mol [9]. At 350 °C, EA melts quickly, making it a very thermostable chemical. It is entirely soluble in caustic potash, completely soluble in alcohol, and sparingly soluble in ether. It is insoluble in water. Because of its structural makeup, which includes 2 hydrocarbon rings, 2 lactones, and 4 hydroxyl groups, EA can take up electrons from different sources and serve as an antioxidant [10]. The two pairs of hydroxyl groups in EA are primarily responsible for its antioxidant properties. The compound's two aromatic rings contribute to its antioxidant capacity but decrease its water solubility [11]. EA scavenges free radicals, boosts antioxidant enzymes, lowers ROS generation, and prevents lipid peroxidation [12]. Like most critical vitamins, EA's inherent antioxidant behaviour is mainly related to its ability to scavenge free radicals [13].

EA has been studied to exhibit strong antioxidant potential. Therefore, we further extend the study to evaluate the effect of this polyphenol on the transporters of erythrocyte membrane as a function of aging in humans. Here, the authors studied the effect of EA on the oxidative stress biomarkers in different age groups of red blood cells. EA has been utilised extensively in therapeutics because of its anti-oxidant qualities and capacity to treat illnesses. Numerous illnesses can arise because the body produces free radicals. Thus, using EA in therapeutics may aid in healthy aging by reducing free radicals and preventing oxidative cell damage. This flavonoid was chosen because of its structural property of proton donation, which can potentially change the activity of ion transporters in cell membranes. EA offers protection against oxidative stress and is very effective in scavenging reactive oxygen species (ROS). Additionally, it has demonstrated anti-mutagenic and anti-cancerous properties [14]. EA modifies cellular responses by directly regulating enzyme and ion-transporter activity or by modifying the fluidity of the membrane [10]. However, the precise mechanism underlying the health-promoting benefits of EA is still unknown Fig. 1.

Fig. 1.

Structure of gallic acid and ellagic acid. The structural makeup of ellagic acid consists of 2 hydrocarbon rings, 2 lactones, and 4 hydroxyl groups. The two pairs of hydroxyl groups in ellagic acid are primarily responsible for its antioxidant properties

Research has indicated that oxidative stress is one of the leading causes of changes to membrane transporters' properties. The survival and functions of the cell depend on the plasma membrane. Damage to membrane transporters results in modifications to ion homeostasis, which in turn causes several chronic illnesses, including cancer, diabetes, neurodegenerative diseases, and aging [15]. The simplest model available for examining changes at the cellular level is that of red blood cells and their membrane. Numerous physiochemical alterations that occur in red blood cells as they age have been investigated. It has also been noted that as erythrocytes age, their cell density increases, and their cell volume decreases [16]. The membrane's NHE system, which is amiloride-sensitive and electroneutral, regulates acidosis by limiting the entry of Na⁺ and the removal of H⁺ [15]. Almost every live cell has a tightly linked exchange of Na⁺ for H⁺ across its surface membrane. Because it catalyses the electro-neutral exchange of external sodium and internal hydrogen, the Na⁺, H⁺ exchanger is widely distributed in the plasma membrane of red blood cells and is essential for maintaining intracellular pH and cell volume homeostasis [17]. The transmembrane proteins Na⁺, K⁺ ATPase control the amounts of Na⁺ and K⁺ ions linked to ATP hydrolysis intracellularly and extracellularly. This enzyme is responsible for the active passage of K⁺ and Na⁺ ions through the cell membrane. Muscles, brain, and erythrocytes have high quantities of the enzymes Na⁺, K⁺ ATPase [18]. Ca²⁺ ATPase control Ca²⁺ ion homeostasis, which is essential for several cellular processes. Reactive oxygen species attack the amino acid residues, generating carbonyl moieties. These are used to quantify the damage to proteins and are thought to be an early sign of protein oxidation. [19]. The membrane's oxidative damage leads to the loss of some specific protein functions. The oxidatively damaged erythrocyte membrane significantly affects its mechanical properties [20]. The accumulation of the carbonyl group is considered an indicator of oxidative stress in humans during aging [21].

The effects of oxidative stress in different cells and tissues are mitigated by antioxidants derived from diverse plant sources. In this work, the authors aim to demonstrate that EA is a strong antioxidant that inhibits NHE activity and increases the activity of Na⁺, K⁺, and Ca²⁺ ATPases. The current investigation assessed the dose-dependent impact of EA on human erythrocyte membrane transporters under both experimentally produced oxidative stress and control settings. Here, we describe the aging-related alterations in human erythrocyte membranes. In our work, we have clarified the protective role of EA on oxidative stress indicators during aging. We looked further into the effects of EA on NHE activity, Na⁺, K⁺ ATPase, Ca²⁺ATPase, a protein carbonyl group, and osmotic fragility during human aging based on studies on the effects of a few dietary polyphenols on membrane-bound ATPases, NHE activity, and osmotic fragility.

Material and Methods

Chemicals and Reagents

The following chemicals have been used for experimental purposes: Ellagic acid, amiloride hydrochloride, ouabain, bovine serum albumin, 2,4-dinitrophenylhydrazine, guanidine hydrochloride, 4,4-diisothiocyanatostilbene-2,20-disulphonic acid, and ATP. The best analytical grade chemicals were acquired from HIMEDIA labs and Sigma Aldrich. A UV–VIS spectrophotometer was used to carry out the spectrophotometric analysis.

Samples

Standard, healthy volunteers of both genders, aged 18–60 years and above (n = 80), were included in the study. Three age groups made up the human subjects: young age group that was < 35 years (n = 35), middle age group between 36 to 60 years (n = 25), and old age group > 60 years (n = 25). The selection criteria that were used were as outlined by Maurya and Rizvi [20]. The samples were collected from the Haryana region. Exclusion criteria include (a) none of the subjects was under any medication, (b) no pregnant and lactating women were included in the study, (c) none of the subjects were smokers and (d) none of the subjects was screened for any significant illness or any sort of acute or chronic common medical conditions. The blood collection protocol was carried out by the standards set forth by the institutional ethics committee (Ref no. CUH/IHEC/2021/03).

Blood Isolation and Red Blood Cell Membrane Preparation

Fresh blood was drawn by arm venipuncture and centrifuged at 1800 g for 10 min at 4 °C to separate plasma and packed red blood cells (PRBCs). The supernatant containing plasma was extracted and pipetted out. The top 10–25% layer and the buffy coat were thrown away. Using an isotonic solution (phosphate saline buffer) of pH 7.4, the packed red blood cells obtained as pellets were washed twice or thrice. The red blood cell membrane was separated using the osmotic shock technique given by Marchesi and Palade [22]. Protein quantification was carried out using the method provided by Lowry, Rosebrough [23].

Estimation of Na⁺, K⁺Exchanger Activity

The Na⁺, K⁺ exchanger activity was determined as described by Matteucci, Rizvi [24]. Spectrophotometric determination of the NHE activity is based on the variation in the acidification rate of the medium with and without amiloride. Briefly, packed red blood cells were dissolved in a mixture containing MgCl₂ (1 mM), KCl (1 mM), NaCl (150 mM), and glucose (10 mM) and stirred magnetically for approximately 5 min at 37 °C. The pH of the suspension was brought to a range of 6.35–6.45 within a period of 10 min by adding HCl (0.2 M) prepared in NaCl (150 mM). Thereafter, by adding 0.2 M DIDS the pH was brought in the range of 7.95–8.00 by using NaOH (0.05 M) prepared in NaCl (150 mM). A parallel experiment introduced DIDS containing amiloride and subsequently, the proton's outflow during the initial minute was noted. At 37 °C, NHE activity was measured regarding proton efflux mmol/L RBC/hour.

Estimation of Na⁺, K⁺ATPase Activity

Na⁺, K⁺ ATPase activity was determined as described earlier by Suhail and Rizvi [25]. The activity was determined spectrophotometrically by measuring inorganic phosphate (Pi) released because of ATP hydrolysis in the presence and absence of ouabain (an enzyme inhibitor). The final assay mixture contained membrane protein NaCl (140 mmol/L), KCl (20 mmol/L), MgCl₂ (3 mmol/L), and imidazole (30 mmol/L) at pH 7.25 and incubated with or without ouabain for 30 min at optimal temperature. The parallel experiment was conducted by adding ATP (6 mmol/L). After around 30 min, the incubation was stopped by adding 3.5 mL of a solution that included SDS (2%), ammonium molybdate (0.5%), and H₂SO₄ (0.5 mol/L). The activity of Na⁺, K⁺ ATPase was measured in terms of μmol Pi released/mg protein/hour at 37 °C.

Estimation of Ca²⁺ATPase Activity

Ca²⁺ ATPase activity was determined as described earlier by Pandey, Jha [26]. Briefly, the mixture consisted of an erythrocyte membrane incubated with a solution containing NaCl (80 mmol/L), EGTA (0.1 mM), MgCl₂ (3 mM), KCl (15 mM), Tris–HCl (50 mM, pH 7.4), and ouabain (0.5 mM) in the presence and absence of CaCl₂ (0.2 mM) at optimal temperature for 30 min. To initiate the reaction ATP (6 mM) was added. Calmodulin (40 units/mL), was added to the reaction mixture. After the incubation period, a cold solution containing H₂SO₄ (0.4 M), ammonium molybdate (0.5%), and sodium dodecyl sulphate (2%) was added to halt the reaction. 0.04 mL of a solution containing sodium sulfite (1.2%), sodium metabisulfite (1.2%), and 1-amino-2-naphthol-4-sulphonic acid (0.2%) was added to each tube and shaken for 10 min, followed by a centrifugation step. O.D was measured at 650 nm, and Ca²⁺ATPase activity was measured in μmol Pi/mg protein/hour.

Estimation of Protein Carbonyl Group

Carbonyl group content was evaluated by the method described by Reznick and Packer [27]. 200 μL of isolated membrane samples dissolved in phosphate buffer saline were taken in two different test tubes labelled as control and test. 10 mM 2,4-dinitrophenylhydrazine was prepared in 2 M HCl and dissolved only in the tube labelled as a test, in the control tube, 4 M HCl was added alone without 2,4-dinitrophenylhydrazine. The contents were vortexed properly and kept for 1 h at optimal temperature. To facilitate proper reaction with the protein, the tubes were shaken every 10 min. After an hour of incubation, trichloroacetic acid (20%) was added to each tube and kept in ice for about 10 min, followed by a centrifugation step to obtain protein pellets. The pellets obtained were washed 2–3 times using 1:1 (v/v) ethanol: ethyl acetate. This step removed the leftover lipid remnants and 2,4-dinitrophenylhydrazine that remained unreacted. At last, 6 M guanidine hydrochloride was dissolved in protein pellets and left at optimal temperature for 10 min, followed by centrifugation and thereafter O.D was measured at 370 nm. The protein carbonyl content was calculated using an absorption coefficient of 22000 M⁻¹ cm⁻¹.

Estimation of Osmotic Fragility

Osmotic fragility was assessed as the method given by Fujii [28]. Different range of NaCl solutions (0.1–0.9%) were used to carry out the study. This gradient was used to observe initial hemolysis to complete hemolysis. 100 μL of PRBCs were mixed with 5 mL of NaCl concentrations in other tubes. The mixture was correctly mixed without vortexing. The tubes were incubated for an hour at room temperature. The mixture was then centrifuged at 2500 g for 5 min. The absorbance of the supernatant was read at 540 nm. The osmotic fragility of the erythrocyte is used as a measure of the susceptibility of RBC to haemolysis. Distilled water used as a control showed complete lysis, and 0.9% NaCl solution showed no lysis. The per cent haemolysis was calculated using the following equation: % haemolysis = (OD sample)/(OD of distilled water) × 100 [29].

Investigations Using Ellagic Acid and Inducing Oxidative Stress

Before the assay, erythrocyte ghosts (0.8–1.5 mg) were incubated with EA at several doses in PBS (pH 7.4) for one hour at 37 °C. This allowed researchers to examine the effect of EA on erythrocyte membrane transporters. Oxidative stress was created in vitro by treating erythrocyte ghosts with 10⁻⁵ M t-BHP for one hour at 37 °C. Erythrocyte ghosts were co-incubated with t-BHP and EA for an hour at 37 °C with light shaking to examine the impact of EA. After incubation, membrane transporter activities were measured, and further, the protein carbonyl group and osmotic fragility were studied.

Molecular Docking

Using PDB files for the transporters and ligand, ellagic acid, in MOL2 format, the various transporters found on the erythrocyte membrane were molecularly docked. The Zhang lab's Edock was used for the docking (https://zhanglab.dcmb.med.umich.edu/EDock/) [30]. To view the final structural model, the docked model (the top model of each docking) was loaded into UCSF Chimera 1.17.3. [31].

Statistical Analysis

The Windows 8.00 version of GraphPad Prism was utilized for all statistical analyses. Student's t-test was used to assess statistical differences and analyze the effect of EA in different age groups and a difference was deemed significant if its p-value was less than 0.05.

Results

We calculated the effect of EA administration, with a final concentration ranging from 10⁻⁵–10⁻⁷ M, on Na⁺, K⁺ exchanger activity in red blood cells of age groups ranging from 18 years to above 60 years as shown in Fig. 2. We note that the NHE activity increases relative to their respective controls with increasing age in each group. Inducing stress significantly increases (p < 0.001) Na⁺, K⁺ exchanger activity in young, middle, and old age groups, when compared to their respective controls. The administration of various doses of EA further reduces NHE activity. NHE activity in the younger age group is substantially lower than that of stress-induced red blood cells at 10⁻⁵ M concentration EA (p < 0.001). In younger age groups, a significant decline in Na⁺, K⁺ exchanger activity was also observed with t-BHP-induced stressed cells at 10⁻⁶ M and 10⁻⁷ M concentrations of EA. Comparing the middle-aged participants to the t-BHP-induced red blood cells in the same age group, there was a substantial decline (p < 0.001) in NHE activity at different EA doses. Similar findings have been noted in elderly persons, with a significant (p < 0.01) drop in NHE activity compared to stressed cells within a similar age group. We further deduce from Fig. 2 that NHE's decreased activity is concentration-dependent. Different age groups exhibit more significant NHE activity as the concentration of EA is lowered.

Fig. 2.

Effect of ellagic acid administration (10⁻⁵–10⁻⁷ M final concentration) on red blood cells NHE activity in young (< 35 years; n = 35), middle (36–60 years; n = 25) and old (> 60 years; n = 20) age group. NHE activity was expressed in terms of micromole of Pi released/hr/mg membrane protein at 37 °C. Data are expressed as mean ± SD. *, p < 0.001 compared to t-BHP treated with respective control; #, p < 0.001 compared to t-BHP with EA treated in young age group; $, p < 0.001 compared to t-BHP with EA treated in middle age group; @, p < 0.01 compared to t-BHP with EA treated in old age group. M, molar

Figure 3 illustrates how we calculated the effects of EA administration, at concentrations ranging from 10⁻⁵–10⁻⁷ M, on the activity of Na⁺, K⁺ ATPase in red blood cells in the young, middle, and elderly age groups. There is a decline in p value (p < 0.001) in Na⁺, K⁺ ATPase activity of the membrane of red blood cells when comparing middle-aged and older human participants to a younger group of the control samples. A noteworthy reduction (p < 0.001) was noted in the activity of Na⁺, K⁺ ATPase in the stress-induced membrane of red blood cells when compared to the corresponding control of all the age groups. When oxidative damage is generated by t-BHP activity of Na⁺, K⁺ ATPase further declines; however, administration of 10⁻⁵ M EA upregulates Na⁺, K⁺ ATPase in the control sample, which is higher in the subjects of age ranging between < 18 and 35 years of age. The effect of 10⁻⁷ M EA is negligible in younger age groups when contrasted with the strained erythrocyte membrane caused by t-BHP. Higher concentrations of EA, such as 10⁻⁵ M, increase activity of Na⁺, K⁺ ATPase when compared to reduced concentrations, such as 10⁻⁶ M and 10⁻⁷ M, as seen in all age groups. Except for the young age group, Na⁺, K⁺ ATPase activity rises in a concentration-dependent way compared to t-BHP-induced erythrocyte membrane. The activity is noticeably more substantial at 10⁻⁵ M concentrations than at 10⁻⁶ M and 10⁻⁷ M values. A dose-dependent reduction in Na⁺,K⁺ ATPase activity was seen when EA was administered. Compared to erythrocyte membrane induced with t-BHP, EA treatment had a greater impact on Na⁺, K⁺ ATPase activity (p < 0.001) in younger age groups. Participants' Na⁺, K⁺ ATPase activity is significantly higher (p < 0.001) than that of the t-BHP-induced strained erythrocyte membrane belonging to middle-aged groups following the administration of EA. Compared to stress-induced red blood cell membranes of elderly groups, the Na⁺, K⁺ ATPase activity likewise increased significantly (p < 0.001) in membranes treated by EA. In human participants of various age groups, the activity of Na⁺, K⁺ ATPase of the membrane was considerably reduced (p < 0.001).

Fig. 3.

Effect of ellagic acid administration (10⁻⁵–10⁻⁷ M final concentration) on red blood cells Na⁺, K⁺ ATPase activity in young (< 35 years; n = 35), middle (36–60 years; n = 25) and old (> 60 years; n = 20) age group. Na⁺, K⁺ -ATPase activity was expressed in terms of micromole of Pi released / hr/mg membrane protein at 37 °C. Data are expressed as mean ± SD. *, p < 0.001 compared to t-BHP treated with respective control; #, p < 0.001 compared to t-BHP with EA treated in young age group; $, p < 0.001 compared to t-BHP with EA treated in middle age group; @, p < 0.001 compared to t-BHP with EA treated in old age group. M, molar

As illustrated in Fig. 4, we calculated the effect of EA administration (10⁻⁵–10⁻⁷ M final concentration) on Ca²⁺ ATPase activity of the membrane of red blood cells in the young, medium, and elderly age groups. The activity of Ca²⁺ ATPase in the control sample is elevated in the subjects belonging to the younger age groups; Ca²⁺ ATPase further reduces when oxidatively stressed via t-BHP and Ca²⁺ ATPase activity is upregulated in a concentration-dependent manner when EA of varying doses is administered. Additionally, this has been noted in middle-aged and elderly populations. In middle-aged and older human participants, the erythrocyte membrane Ca²⁺ ATPase activity dramatically declines (p < 0.001) compared to younger ones. In all age groups, there was a noteworthy reduction (p < 0.001) in the Ca²⁺ ATPase activity in the t-BHP-induced erythrocyte membrane compared to the corresponding control. Ca²⁺ ATPase activity is increased by higher concentrations of EA (10⁻⁵ M) in comparison to lower concentrations (10⁻⁶ M and 10⁻⁷ M), as was seen in all age groups. In a dose-dependent manner, Ca²⁺ ATPase activity rises compared to t-BHP-induced erythrocyte membrane. The activity is noticeably more potent at 10⁻⁵ M concentrations than 10⁻⁶ M and 10⁻⁷ M values. The administration of EA was found to cause a concentration-dependent reduction in Ca²⁺ ATPase activity. Compared to erythrocyte membrane treated with t-BHP, the effect of EA administration on Ca²⁺ ATPase activity was more substantial (p < 0.001) in younger age groups. Compared to the same age group's strained erythrocyte membrane caused by t-BHP, the increase in Ca²⁺ ATPase activity in participants of middle-aged groups is less substantial (p < 0.01). Additionally, erythrocyte membranes given with EA showed a significant (p < 0.001) increase in Ca²⁺ ATPase activity compared to those caused by stress in the older age group.

Fig. 4.

Effect of ellagic acid administration (10⁻⁵–10⁻⁷ M final concentration) on red blood cells Ca²⁺ ATPase activity in young (< 35 years; n = 35), middle (36–60 years; n = 25) and old (> 60 years; n = 20) age group. Ca²⁺ ATPase activity was expressed in terms of micromole of Pi released/hr/mg membrane protein at 37 °C. Data are expressed as mean ± SD. *, p < 0.001 compared to t-BHP treated with respective control; #, p < 0.001 compared to t-BHP with EA treated in young age group; $, p < 0.01 compared to t-BHP with EA treated in middle age group; @, p < 0.001 compared to t-BHP with EA treated in old age group. M, molar

Figure 5 depicts the content of carbonyl groups in the erythrocyte membrane in young, middle, and old age groups. The result shows that in the control group, the content of the carbonyl group in the younger age group is lower than that of the middle and old age groups. t-BHP-induced oxidative stress (10⁻⁵ M) significantly increases the protein carbonyl group's content in each age group. Administration of different concentrations of EA (10⁻⁵–10⁻⁷ M) decreases carbonyl group content significantly compared to their respective control in each age group in a concentration-dependent manner.

Fig. 5.

Effect of ellagic acid administration (10⁻⁵–10⁻⁷ M final concentration) on red blood cells protein carbonyl content in young (< 35 years; n = 35), middle (36–60 years; n = 25) and old (> 60 years; n = 20) age group. The protein carbonyl group content in the membrane was expressed in terms of nmol/g of protein. Data are expressed as mean ± SD. #, p < 0.05 compared to the control of the middle and old concerning the young age group; $, p < 0.05 compared to t-BHP induced oxidative stress of middle and old age group concerning the younger age, *, p < 0.05 compared to the control of each group with different concentrations of ellagic acid (10⁻⁵–10⁻⁷ M). M, molar

Figure 6a, b, and c depicts the osmotic fragility in young, middle, and old age groups. The result obtained in all the age groups shows the decreasing lysis of haemoglobin at higher % of NaCl concentrations. The lysis % is highest at 0.1% concentration of NaCl. At 0.9% concentration of NaCl, the lysis percentage is approximately 0. When the native RBCs were induced with stress using t-BHP, the lysis % increased, and after the treatment of EA, the lysis % was found to be decreased in all the age groups.

Fig. 6.

Effect of ellagic acid on osmotic fragility. a The effect of ellagic acid (10⁻⁵ mol/L final concentration) on tert-butyl hydroperoxide (t-BHP) (10⁻⁵ mol/L) induced changes in osmotic fragility in the young age group. Values represent mean ± S.D. b Effect of ellagic acid (10⁻⁵ mol/L final concentration) on tert-butyl hydroperoxide (t-BHP) (10⁻⁵ mol/L) induced changes in osmotic fragility in middle age group. Values represent mean ± S.D. c The effect of ellagic acid (10⁻⁵ mol/L final concentration) on tert-butyl hydroperoxide (t-BHP) (10⁻⁵ mol/L) induced changes in osmotic fragility in the old age group. Values represent mean ± S.D

It has been predicted that EA interacts with Na⁺, K⁺ ATPases in E1 and E2 state. The most likely interacting residues in the E1 state were 337, 440, 442 and 156. In the binding cavity, it interacts with a short sequence that stretches from 329–332, 403–406, 435–437, 461–463, 504–506, 569–573, 645–651, 670–674, and 690–692 as well. It is significant since the sequence motif, precisely VAV329-332, includes potential Na⁺ sites at Val329 and Val332. Ala330 is a residue that coordinates the E1·3Na and E1·3Na·ATP states. In its E2 form, EA is also expected to interact with K⁺, Na⁺ ATPase. 99, 100, 110, 172, 205, 273, 277, 290, 322, 384, 385, 388, and 389 were the residues that interacted with each other and with minor sequence lengths that ranged from 102–104, 106–108, 166–169, 194–202, and 282–287. Asp811, Thr779, Ser782, Glu786, and Asn783 at site I and Asp811, Glu334, Val332, Val329, Ala330, Glu786 and Asn783 at site II are the identical coordinating residues of K⁺ as those of Na⁺ binding sites I and II in the E1·3Na state. As expected from the docking investigations, EA attaches to Ca²⁺ATPase (PDB ID:5ZTF). Small motifs between 325–327, 485–487, and 595–597 are where EA interacts. EA also interacts with the Ca²⁺ATPase residues at 460,462,467, 530,532, 648, and 676,677 Fig. 7.

Fig. 7.

Represents the docking study of ellagic acid as a ligand with different membrane-bound enzymes as substrates. The molecular docking of different membrane transporters with EA as a ligand has also been performed. The docking of Na⁺, K⁺ ATPases at E1 (PDB ID: 7E1Z) and E2 (PDB ID: 7E20) state, Ca²⁺ATPases (PDB ID: 5ZTF) and Na⁺, H⁺ antiporter (PDB ID: 7B4L) was performed using MOL2 format

Discussion

Through its ability to control the transport of water along with ions via the cell membrane, NHE is a vital regulator of cell volume and intracellular ions in diseased and healthy situations [32]. HNE is a secondary active transporter that controls the volume of the cell, intracellular pH, and Na⁺ concentration. Due to haemoglobin's deoxygenation throughout aging, age-dependent NHE activity has significantly increased in humans and rats. Studies have shown that diabetes mellitus is associated with considerably greater NHE activity [33]. Maintaining the ideal concentration of inorganic anions and cations is the primary job of the cellular membrane. Ionic homeostasis and the ionic gradient are preserved by the membrane transporters and exchangers that are fully functional in the cell membrane. Over the membrane, these transporters and exchangers control physiological processes such as osmotic balance, cytoplasmic pH, cell volume, shape, and growth [34].

Maurya [35] has investigated how EA affects aging human erythrocytes. According to the study, EA reduces the elevated malondialdehyde (MDA) level in the erythrocytes of elderly participants. Membrane-bound ATPases are lipid-derived enzymes that help keep ion gradients in cells intact. Increased oxidative stress has been demonstrated to impact the Na⁺, K⁺ ATPases. Age-related increase in lipid peroxidation of membrane lipids may be connected to decreased human Na⁺, K⁺ ATPase function [26]. As human aging develops, the erythrocyte membrane has a range of responsibilities, from maintaining the adhesiveness of cell development to healing injured tissue. The membrane-bound enzymes, called ATPases, preserve the ionic gradient between the extracellular and intracellular phases. Ca²⁺ATPase and Na⁺, K⁺ ATPase transport Ca²⁺ and Na⁺, K⁺ ions across the cell membrane by means of ATP hydrolysis [36]. The ionic gradient becomes unbalanced due to the ATPases' decreased activity, which further modifies erythrocyte volume, membrane fluidity, and cell shape. In cells, calcium ion control is carried out by Ca²⁺ATPase. A rise in internal calcium levels causes a cell's volume and shape to change, increasing hemolysis and cell stiffness. Na⁺, K⁺ ATPase maintain osmotic balance across the membrane, which controls the volume of erythrocytes and the viscosity of the cytoplasm. Disease pathogenesis results from any reduction in the activity of the Na⁺, K⁺ ATPase at the cellular level [37]. Age-dependent increases in NHE activity and decreases in Na⁺, K⁺ and Ca²⁺ATPases in humans may be protective against increased oxidative stress in individuals [38].

Proteins are highly prone to oxidative stress caused by ROS. These reactive species attack and modify amino acid residues such as histidine, proline, arginine and lysine, generating a carbonyl group. Carbonyl group is a biomarker for oxidative damage of proteins [39]. The increase in the content of the protein carbonyl group is the hallmark of aging. The RBC plasma membrane is composed of a protein-lipid bilayer, which helps to maintain the erythrocyte. The oxidation of the amino acid chains leads to protein damage and, therefore, increased generation of the carbonyl group, which is responsible for many diseases [40]. Osmotic fragility is used to diagnose thalassemia, autoimmune haemolytic anaemia, etc. It is a concentration-based analytical method. The fragility test is used to determine the resistance of erythrocytes to haemolysis and, therefore, is involved in the diagnosis of various disorders [41]. Osmotic fragility depends on the integrity of the cell membrane, the composition of RBCs, and the ratio of volume to surface area. Erythrocytic haemolysis is the process of releasing haemoglobin into the plasma because of membrane rupture. Oxidative stress accelerates erythrocytic fragility [42].

It can be deduced from Fig. 2 that NHE activity rises with age. NHE activity rises greater in the presence of oxidative stress, suggesting that NHE activity could be used to detect oxidative stress in people as they age. NHE activity declines when treated with EA, suggesting that EA plays a part in lowering oxidative stress. The NHE activity rises in all age groups as we reduce the EA concentration from 10⁻⁵ to 10⁻⁶ M and then to 10⁻⁷ M. Dietary flavonoids reduce NHE activity, which is a crucial part of their aging process. After EA is administered, there is a notable reduction in NHE activity, which provides protection against age-related illnesses and aging itself. We can determine from Figs. 3 and 4 that membrane-bound ATPases become less active as humans age. Oxidative stress causes a further decline in the membrane-bound enzyme activity. The activity of membrane-bound enzymes is markedly increased when EA is applied to this erythrocyte membrane. There is a concentration-dependent increase in activity. The enhanced activity is more significant at concentrations of 10⁻⁵ M EA, and the activity of membrane-bound enzymes declines as concentrations are lowered to 10⁻⁶ M and 10⁻⁷ M EA.

The increase in NHE activity with human aging is due to the change in the cell's redox status during aging. A decreased activity of Na⁺, K⁺ and Ca²⁺ATPases with aging has been correlated with an increased level of MDA, a byproduct of lipid peroxidation with human aging [26]. Age-dependent reduction in ATPase activity and increase in NHE activity during human aging may be a compensatory mechanism for adjusting to higher levels of oxidative stress. EA may also function through the phospholipid bilayer, which is an obvious target for ROS due to unsaturated fatty acids. It has been demonstrated that modifications to the membrane's bilayer thickness, fluidity, and head group composition impact the ion transporter's activity. The characteristics of the cell membrane determine the activities of ion transporters, and therefore, the activity of EA may alter the physicochemical characteristics of the membrane.

From Fig. 5, we infer that the content of the protein carbonyl group increases with age in humans. Induced oxidative stress further increases the protein carbonyl group content, and administration of different concentrations of EA decreases protein carbonyl group content significantly in other age groups. The results of our study show that 10⁻⁵ M and 10⁻⁶ M concentrations of EA decrease protein carbonyl group content in each age group significantly compared to their respective controls. 10⁻⁷ M concentration does not show any significant effect on younger and middle-aged groups but shows significant results at an older age compared to the control group. Figure 6a, b, and c shows the osmotic fragility of erythrocytes in different age groups. We used a single concentration (10⁻⁵ M) of EA to study osmotic fragility. The osmotic fragility of the erythrocyte is used as a measure of the susceptibility of RBC to haemolysis. Distilled water used as a control showed complete lysis, and 0.9% NaCl solution showed no lysis.

As depicted in Fig. 7, molecular docking of different membrane transporters with EA as a ligand has also been performed. The docking of Na⁺, K⁺ ATPases at E1 (PDB ID: 7E1Z) and E2 (PDB ID: 7E20) state, Ca²⁺ATPases (PDB ID: 5ZTF) and Na⁺, H⁺ antiporter (PDB ID: 7B4L) was performed using MOL2 format.

Limitations and Future Perspectives

The study presented here is novel in the context of EA, but it still has some limitations. The sample size of the study is limited and confined to a particular region. Even though the individuals selected were devoid of any sort of medications, their diet pattern was not considered. The protective effects of EA in various disorders have been reported in some studies, but not much is known about the role of EA in human aging, particularly in the membrane transporters of red blood cells. The authors believe that the study presented here is novel and will further pave the way for research related to human aging.

Conclusion

This work shows that EA modulates the activity of membrane-bound Na⁺, H⁺ exchangers, Na⁺, K ⁺ATPase, and Ca²⁺ATPase in erythrocytes throughout human aging. EA dramatically reduces the activity of membrane-bound enzymes in erythrocytes as people age. Our present study highlights the effect of EA on the protein carbonyl content and osmotic fragility in the t-BHP-induced erythrocytes. The impact of EA on the protein carbonyl group and osmotic fragility has been correlated with aging. The results above support the notion that EA is an efficient modulator of Na⁺, H⁺ exchanger, Na⁺, K⁺ ATPase, Ca²⁺ATPase, a protein carbonyl group, and osmotic fragility, preserving membrane integrity as humans age naturally.

Author Contributions

Conceptualization, Writing—original draft preparation: [Deepika]; Supervision [Pawan Kumar Maurya]; Writing—review and editing: [Tikam Chand Dakal], [Richa]. All authors read and approved the final manuscript.

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript.

Availability of Data and Materials

Not applicable.

Declarations

Conflict of interests

The authors have no relevant financial or non-financial interests to disclose.

Ethical Approval

The institutional ethical committee has approved this study by its guidelines (Ref no. CUH/IHEC/2021/03).

Human Ethics and Consent to Participate Declarations

Informed consent was obtained from all individual participants included in the study.