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Abstract

Industrial apple pomace, a biowaste generated during apple processing, is rich in cell wall polysaccharides and phenolics. These biologically active compounds are reported to be highly beneficial from the nutritional and health point of view. In the present study, the total phenolic content in the apple pomace aqueous extract (APE) was estimated and evaluated for its possible antioxidant and hepatoprotective efficacy in carbon tetrachloride (CCl₄)-induced liver injury mice model. The aqueous extract exhibited 2,2-diphenyl-2-picrylhydrazyl free radical scavenging activity in vitro. Under in vivo study, mice were treated with APE (200 mg and 400 mg/kg body weight) for 2 weeks prior to the administration of CCl₄ (30% v/v). The serum liver injury markers alanine transaminase, aspartate transaminase, and alkaline phosphatase were significantly lowered by APE in a dose-dependent manner. The levels of antioxidant parameters superoxide dismutase (SOD), reduced glutathione (redGSH), and lipid peroxidation were also improved by APE in liver homogenate. Histopathological studies revealed that APE treatment significantly lowered the CCl₄-induced necrotic changes in the liver. Furthermore, terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end-labeling assay showed that CCl₄-induced apoptosis in the liver was significantly inhibited by APE in a dose-dependent manner. Immunohistochemistry results showed higher expression of nuclear erythroid 2-related factor 2 (Nrf2) in the liver of the APE-treated mice, a key regulator of antioxidative response. In conclusion, the results of the present study revealed the hepatoprotective efficacy of APE by inhibiting CCl₄-induced apoptosis, which is due to its antioxidant activity and the ability to induce Nrf2 protein expression.

Keywords

Apple pomace hepatoprotective apoptosis Nrf2

Introduction

Liver being an essential organ in the body plays critical role in different metabolic reactions, biotransformation, and detoxification of harmful substances, for example, toxic industrial chemicals, water pollutants, free radicals, and so on.¹ Changing life style, food habits, presence of xenobiotics in the environment, and many drugs (like statins and acetaminophen) cause asymptomatic elevation of liver injury marker enzymes, thereby leading to oxidative stress and hepatotoxicity. Oxidative stress is regarded as a mediator of acute and chronic liver injury. Carbon tetrachloride (CCl₄)-induced injury to liver is the most extensively studied system for oxidative hepatotoxicity.² CCl₄ injury is mediated by reactive metabolite-trichloromethyl free radical $(^{\cdot} {CCl}_{3})$ formed by hemolytic cleavage of trichloromethyl peroxy free radical (Cl₃COO⁻) by cytochrome P450 of liver cells_. ³ Cytochrome P450 2E1 (CYP2E1) is known to be associated with metabolism of CCl₄ and associated toxicity.⁴ CCl₄-induced damage at molecular level has been studied by the expression of oxidative stress-mediated transcription factors nuclear factor κB, peroxisome proliferator-activated receptor β/δ, nuclear erythroid 2-related factor 2 (Nrf2),⁵ and upregulation of apoptotic genes via induction of Jun amino-terminal kinase signaling.⁶ The formed free radicals combine with membrane lipids and leads to peroxidation and eventually cell necrosis.⁷ The scavenging of free radicals and activation of antioxidant mechanism is important for the protection of liver against hepatic injury.

The Nrf2 acts as key manager of gene regulatory network under stress response and is activated to augment the crowd of antioxidants and drug-metabolizing enzymes. Antioxidant response element (ARE)-associated expression plays important role against chemically induced oxidative stress by activation of protein kinase C, thereby increasing the expression of Nrf2.⁸

Since ancient times, plants have been utilized as a protective agent against several toxicities for being effective and safer. Studies have been carried out in past to show the protective effect of plant extracts against chemical and drug-induced hepatotoxicity.⁹ Dietary polyphenols play an important role in self-defense mechanism against oxidative stress.¹⁰ Apple pomace is a highly nutritious industrial waste and possesses cell wall polysaccharides and dietary polyphenols as main constituents.^11,12 Due to side effects of synthetic drugs, interest of manufacturer and consumer is moving to production of natural products for the prevention of disease. Several reports have been published regarding physiological functions of apple polyphenols, such as antiallergic,¹³ antitumor,¹⁴ and antiobesity effects,¹⁵ as well as inhibitory effects on triglyceride absorption¹⁶ in human studies.

Most of the in vivo studies were focused on establishing the role of phenolics as free radical scavenger. In earlier studies, the antioxidant effect of apple polyphenols¹⁷ was evaluated, but the molecular mechanism was not explored. Therefore, the main aim of this study was to explore the molecular mechanism involved in the hepatoprotective effects of apple pomace extract in CCl₄-induced liver toxicity in mice.

Materials and methods

Chemicals and reagents

Gallic acid, quercetin, trolox, 2,2-diphenyl-2-picrylhydrazyl (DPPH), and aluminum chloride were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Potassium acetate, hydrogen peroxide (H₂O₂), Folin’s reagent, and sodium carbonate of analytical grade were procured from Merck Pvt. Ltd (Germany). Thiobarbituric acid (TBA), nitroblue tetrazolium, nicotinamide adenine dinucleotide, and 5,5′-dithiobis 2-nitrobenzoic acid (DTNB) for enzyme analysis and hematoxylin and eosin for histopathology were purchased from Himedia Labs (Mumbai, Maharashtra, India). CCl₄ was procured from Rankem Chemicals (Gurgaon, Haryana, India). Kits for alanine transaminase (ALT), aspartate transaminase (AST) and alkaline phosphatase (ALP) analysis were purchased from ERBA (Germany) and SOD was obtained from Sigma Aldrich. All the chemicals and solvents were of analytical grade and commercially available.

Raw materials

Industrial apple pomace, a mixture of different cultivars (Red Delicious, Golden Delicious, Royal Delicious, and Red Chief), was collected from Himachal Pradesh Horticultural Produce Marketing and Processing Corporation Ltd, a fruit processing unit located at Parwanoo, Himachal Pradesh, India. The drying of pomace was carried out in industrial tray drying oven (MSW-215, Macro Scientific Works Pvt. Ltd, New Delhi, India) at 60 ± 2°C which then was powdered (1 mm) using cutting mill (Retsh SM 100).

Extract preparation

The extraction of polyphenols from dried apple pomace was done using water as extraction solvent. Accurately weighed 1 kg apple pomace was extracted thrice with water following continuous stirring. The extract was filtered with cotton, followed by Whatman No. 1 filter paper. The filtrates were then pooled and concentrated (between 50°C and 80°C) under vacuum using a rotary evaporator (Buchi 210, Switzerland) and lyophilized (Labconco, Kansas City, Missouri, USA) until constant weight was attained. The lyophilized extract was labeled as apple pomace aqueous extract (APE) and stored at 4°C for further analysis. For spectrophotometric analysis, sample was filtered through 0.45 μm filter (Millipore, Billerica, Massachusetts, USA).

Total polyphenol content

The total phenolic content (TPC) of apple pomace was determined using Folin–Ciocalteu’s method.¹⁸ Results of total polyphenols were expressed as gallic acid equivalent in ^—milligram per gram of apple pomace. The total flavonoid content was quantified by the colorimetric method¹⁹ and was expressed as quercetin equivalent in ^—milligram per gram of apple pomace.

DPPH assay

The antioxidant potential of aqueous extract was evaluated on the basis of its radical scavenging potential by DPPH method.²⁰ Aliquot of 100 µL was added to 2.9 mL of 100 μM DPPH solution. Reaction mixture was vortexed for 1 min and kept at room temperature for 30 min, and absorbance was measured at 517 nm using an ultraviolet–visible spectrophotometer (T 90⁺, PG instruments Ltd, Leicestershire, United Kingdom). The percent inhibition was calculated using the following equation:

% Inhibition = [\frac{(A_{B} - A_{A})}{A_{B}}] \times 100,

where A_A is the absorption of respective extract and A_B is the absorption of blank sample.

The results were expressed as milligram of trolox per gram weight of sample.

HPLC quantification of phenolics

Quantification of individual phenolics was performed using previously developed reversed phase high-performance liquid chromatography (RP-HPLC) method.²¹ The analysis of phenolic constituents was performed using Waters system with an autosampler-2707 and photodiode array (PDA) 2998 detector (Milford, Massachusetts, USA). Separation of phenolics was achieved using Synergi MAX RP80 (Torrance, California, USA), C₁₂ column (4.6×250 mm length, 4 μm particle size). Data were evaluated using Empower software (Waters Corporation). The mobile phase consisted of acetonitrile (solvent A) and 0.01% trifluoroacetic (solvent B). Standard stock solutions were prepared at 1 mg/mL concentration. Calibration curves of all the standards were prepared using 5, 10, 15, 20, and 25 μg/mL concentration. The monitoring wavelength was 280 nm.

Experimental animals

Male Swiss albino mice (20–30 g) were used in the study. They were housed in well-ventilated room at 23 ± 2°C, with a humidity of 65–70%, and a 12-h light/12-h dark cycle. Animals were fed with a standard chow and distilled water ad libitum. The experiment was approved by Institutional Animal Ethics Committee and was performed per Committee for the Purpose of Control and Supervision of Experiments on Animals guidelines.

Liver injury model

The animals were randomly divided into four groups with five animals in each group. The control and CCl₄ groups were fed with distilled water for 14 days. The treatment groups were given 200 mg/kg body weight (BW) and 400 mg/kg BW of APE daily orally for 14 days. On 13th day, CCl₄ (30% v/v) mixed with corn oil was given intraperitoneally (i.p.) to all three groups except normal control.¹⁷ On 14th day, the mice were euthanized after blood collection, and tissue samples were collected for further analysis.

Liver function assays

At the end of experiment, blood sample was collected from each animal via cardiac puncture under light anesthesia and was allowed to clot for serum separation. Serum activities of ALT, AST, and ALP are indicators of liver diseases. The serum was collected, and the activities of liver injury marker enzymes ALT, AST, and ALP were estimated using commercially available ERBA diagnostic kits as reported earlier.²²

Antioxidant assays

After the animals were killed, liver was removed and a section was kept for the biochemical estimations and rest for histopathology. The section for biochemical estimation was homogenized in 0.1 M ice-cold phosphate-buffered saline (PBS) using a homogenizer (IKA T-10 basic). The homogenate was centrifuged for 10 min at 10,000 r/min and supernatant was used for further study. Per manufacturer’s instructions, SOD activity was examined in liver homogenate, and the results were expressed as unit per milligram protein tissue.²³ The nonprotein liver-reduced glutathione (redGSH) levels were measured by the method using DTNB.²⁴ The supernatant was mixed and incubated with 50% trichloroacetic acid (TCA; w/v) for 15 min at room temperature. The reaction mixture was then centrifuged at 3000 r/min for 10 min; supernatant was collected, mixed with 0.01 M DTNB, and read at 412 nm. The protein concentration in homogenate was measured by the method of Lowry et al.²⁵ and bovine serum albumin (1 mg/mL, Calbiochem, Billerica, Massachusetts, USA) was used as standard.

Lipid peroxidation

The liver malondialdehyde (MDA) levels were measured using TBA reaction method by Dawra et al.²⁶ The supernatant was incubated with 10% TCA and centrifuged at 2000 r/min for 10 min. The supernatant was collected and incubated again with 0.67% (w/v) TBA and boiled in water bath for 10 min, was cooled, and the obtained pink color solution was read at 535 nm.

Histopathology

A portion of liver was fixed in 10% neutral-buffered formalin for histopathology. The tissues were processed for dehydration, clearing, and were embedded in paraffin. Using microtome (Finesse me, ThermoScientific, Waltham, Massachusetts, USA), 4–5 μm sections were obtained, deparaffinized, dehydrated, and stained with hematoxylin. Counterstaining was done using eosin, and the slides were examined for CCl₄-induced damage. The necrotic area was calculated in the liver sections of five animals per group using bright field Olympus BX53F microscope and cellSens imaging software (Japan).

Immunohistochemistry

TUNEL assay

For detection of apoptotic cells in the tissues, 4–5 μm paraffin-embedded sections were used. Endogenous peroxidase was blocked by incubation in 1.3% H₂O₂ in PBS before enzymatic labeling. The fragmented DNA was end labeled with horseradish peroxidase (HRP) conjugated-biotinylated nucleotide at 3′-OH end using the terminal deoxynucleotidyltransferase recombinant enzyme according to the manufacturer’s instructions (Promega, Madison, Wisconsin, USA). Detection was carried out using H₂O₂ and diaminobenzidine (DAB) substrate. The quantification was done by counting the number of apoptotic nuclei in 10 different microscopic fields/group/five animals under Olympus BX53F bright field microscope.

Nrf2 expression

Nrf2 signaling is known to play a crucial role in the suppression of oxidative stress, hence its role was studied for the observed antioxidant responses of APE during CCl₄ liver injury. The 4–5 μm paraffin-embedded liver sections were mounted, deparaffinized, and hydrated. Antigen retrieval was done by exposing cells to sodium citrate buffer treatment. Quenching of endogenous peroxidases was done using BLOXALL blocking solution (ImmPRESS excel staining kit, Vector Laboratories, Burlingame, California, USA), and exposed sites were blocked by incubating sections with 2.5% (v/v) normal horse serum for 20 min in humidified chamber. Monoclonal Nrf2 raised in rabbit immunoglobulin G primary antibody diluted 1:200 in PBS was used for the study and incubated overnight. Repeated PBS wash was given to remove unbound primary antibody, followed by HRP-conjugated secondary anti-rabbit antibody incubation. The sections were rinsed twice and incubated with DAB substrate. The percentage of Nrf2-positive cells was calculated by counting the positive cells per 10³ cells/group/five animal. Observations were done under Olympus BX53F bright field microscope.

Statistical analysis

The results were expressed as mean ± standard error mean and analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. The results with level of significance p < 0.05 were considered as significant.

Results

Total polyphenol and flavonoid content estimations

The total phenolic content in apple pomace was calculated from the regression equation (y = 0.0024x + 0.0285, R² = 0.9981) and expressed as milligram per gram gallic acid equivalent. Similarly, the flavonoids content in apple pomace were expressed in terms of milligram per gram quercetin equivalent and obtained from regression equation y = 0.005x + 0.1478, R² = 0.9919. The results showed that apple pomace contains 1.12 ± 0.08 mg/g phenolic compounds and 0.281 ± 0.026 mg/g of total flavonoids (Table 1).

Table 1.

Total polyphenol, flavonoid, antioxidant activity, and individual phenolics in apple pomace.

Parameters	AP
Total polyphenol content (mg/g)	1.12 ± 0.08
Total flavonoid content (mg/g)	0.28 ± 0.02
Antioxidant activity (mg/g)	1.52 ± 0.09
Phloretin (µg/g)	0.15 ± 0.09
Epicatechin (µg/g)	2.48 ± 0.07
Coumaric acid (µg/g)	1.57 ± 0.01
Phloridzin (µg/g)	3.55 ± 0.05
Chlorogenic acid (µg/g)	0.65 ± 0.02

AP: apple pomace.

Free radical scavenging activity

A number of spectrophotometric methods are used for the quantification of antioxidant activity, but DPPH is the most acceptable method. Free radicals scavenging activity was estimated by DPPH assay and expressed as trolox equivalent using regression equation (y = 0.1467x + 0.1269, R² = 0.9959). The trolox equivalent antioxidant capacity value of the APE was recorded to be 1.52 ± 0.090 mg trolox/g (Table 1).

RP-HPLC quantification of polyphenols

In the extract, variable amount of phenolic constituents was recorded by RP-HPLC quantification. Results showed the presence of phloridzin, epicatechin, coumaric acid, chlorogenic acid, and phloretin in APE following descending order. Accordingly, RP-HPLC chromatogram showed the presence of quantified phenolic constituents in industrial pomace extract (Figure 1).

Figure 1.

HPLC profile of the aqueous extract of apple pomace. HPLC: high-performance liquid chromatography.

Effect of APE on liver injury markers

CCl₄ administration significantly (p < 0.05) elevated the levels of liver marker enzymes in the serum in CCl₄-intoxicated vehicle control group in comparison with the normal control (Table 2). APE treatment significantly (p < 0.05) lowered the ALT levels at 400 mg/kg dose in comparison with vehicle control group. However, at lower dose, insignificant decline in the ALT levels was observed. APE treatment significantly (p < 0.001) attenuated the AST activity at lower (p < 0.05) as well as higher dose (p < 0.001) in comparison with vehicle control group. Significant (p < 0.05) decrease in ALP activity was observed in the animals treated with higher dose of APE. Again, at lower dose only nonsignificant improvement was observed in comparison with CCl₄ alone group. (Table 2).

Table 2.

Serum biochemical estimations and tissue oxidative damage in different treated groups of mice.^a

Group	ALT (IU/L)	AST (IU/L)	ALP (IU/L)	SOD (U/mg protein)	redGSH (nM/g of wet tissue)	MDA (nM/g of wet tissue)
Control	27.1 ± 1.8^b	32.0 ± 3.3^c	36.2 ± 1.3^b	26.47 ± 1.8^d	57 ± 2.1^d	2.4 ± 0.2^b
CCl₄	48.0 ± 2.8^d	114.8 ± 7.9^d	80.8 ± 7.0^d	8.97 ± 0.5^c	41.4 ± 1.2^b	4.13 ± 0.2^d
CCl₄ + 200 mg/kg APE	39.6 ± 4.5^b,d	78.3 ± 4.4^b	59.7 ± 7.5^b,d	16.21 ± 1.2^b	50.6 ± 1.7^d	2.65 ± 0.4^b
CCl₄ + 400 mg/kg APE	33.3 ± 3.5^b	57.0 ± 5.1^b	48.4 ± 4.7^b	17.99 ± 1.2^b	52.4 ± 1.7^d	2.5 ± 0.1^b

ALT: alanine transaminase; AST: aspartate transaminase; ALP: alkaline phosphatase; ANOVA: analysis of variance; SOD: superoxide dismutase; redGSH: reduced glutathione; MDA: malondialdehyde; CCl₄: carbon tetrachloride.

^aValues within columns (between groups) with different superscripts (b to c) are significantly different by ANOVA (p ≤ 0.05). Data are presented as mean ± SE.

Effect of APE on antioxidant parameters

CCl₄ treatment significantly (p < 0.001) decreased the levels of SOD and redGSH in the liver tissue as compared to the control group (Table 2). APE pretreatment significantly (p < 0.05) improved the SOD levels at 200 as well as 400 mg/kg dose in comparison with the vehicle control. The redGSH levels were also improved significantly (p < 0.05) by APE treatment at lower as well as higher dose in comparison with the vehicle control group. Although the higher dose of APE produced more prominent effect on SOD and redGSH levels, the effect was not found significant in comparison with lower dose (Table 2).

Effect of APE on lipid peroxidation

MDA is the marker of lipid peroxidation and end product of oxidative decomposition of polyunsaturated fatty acid. The effects on the levels of MDA in the liver of different groups are shown in Table 2. CCl₄ significantly increased the hepatic MDA levels in CCl₄ group. The APE treatment at higher as well as lower doses significantly (p < 0.05) lowered the MDA levels in the liver tissue in comparison with CCl₄ alone group.

Effect of APE on histopathology of liver

Histopathology of the control group showed normal hepatic architecture with hepatocytes and central vein. In vehicle control group, liver sections showed nuclear pyknosis in hepatocytes, degeneration of hepatic cord, leukocyte infiltration, and marked periportal necrosis (Figure 2(b)). APE pretreatment at 200 and 400 mg/kg dose showed reduction in leukocytic infiltration and significantly lowered the area of periportal necrosis in comparison with CCl₄ alone group (Figure 2(c) and (d)). The results also indicated that APE alleviated the CCl₄-induced necrosis in the liver in dose-dependent manner.

Figure 2.

Effect of APE on CCl₄-induced alteration in liver histology (magnification ×200). (a) Liver section from control group showing normal liver architecture; (b) liver section from CCl₄ group showing periportal necrosis (arrow); (c, d) liver section from CCl₄ plus 200 mg/kg and 400 mg/kg APE group showing less necrotic changes (arrows); and (e) graphical representation of necrotic area in different groups. *p < 0.05: significant as compared to CCl₄ group. APE: apple pomace aqueous extract; NN: no necrosis observed; CCl₄: carbon tetrachloride.

Effect of APE on apoptosis inhibition

Apoptosis in the liver tissues was assessed by in situ apoptosis detection kit, and the apoptotic index was calculated in the different groups (Figure 3((a) to (e)). The normal control group showed no apoptotic cells, whereas CCl₄ treatment caused marked apoptosis in vehicle control group. The APE at 200 and 400 mg/kg significantly (p < 0.001) lowered the number of apoptotic cells in the liver tissue in comparison with the vehicle control group. The inhibition of apoptosis in the liver by APE was found to be dose dependent, as higher dose significantly (p < 0.05) lowered the apoptotic index (57.8%) in comparison with lower dose (38.3%).

Figure 3.

Effect of APE on CCl₄-induced apoptosis in liver (magnification ×200). (a) Control group; (b) CCl₄ group; (c) CCl₄ plus 200 mg/kg APE group; (d) CCl₄ plus 400 mg/kg APE group; and (e) comparative apoptotic cells in different groups. *p < 0.05: significant as compared to CCl₄ alone group; #p < 0.05: significant as compared to CCl₄ group and 200 mg/kg group. APE: apple pomace aqueous extract; CCl₄: carbon tetrachloride; NAD: no/rare apoptotic cells.

Effect of APE on Nrf2 expression in liver

The hepatic expression of Nrf2 is shown in Figure 4((a) to (d)). The liver sections of the vehicle control group showed reduced expression of Nrf2. However, APE treatment showed significant (p < 0.001) induction of Nrf2 expression in the hepatocytes and Kupffer cells in a dose-dependent manner in comparison with the vehicle control group. At higher dose of APE, most of the immunopositivity for Nrf2 was observed in the nucleus, indicating activation of Nrf2 and its subsequent nuclear translocation.

Figure 4.

Effect of APE on Nrf2 expression in CCl₄-induced liver injury (magnification ×200). (a) Control group; (b) CCl₄ alone group; (c) CCl₄ plus 200 mg/kg APE group showing Nrf2 expression; (d) CCl₄ plus 400 mg/kg APE group showing increased Nrf2 expression; and (e) comparative Nrf2 expression in different groups. *p < 0.001: significant as compared to CCl₄ group; #p < 0.001: significant as compared to CCl₄ group and 200 mg/kg group. Nrf2: nuclear erythroid 2-related factor 2; APE: apple pomace aqueous extract; CCl₄: carbon tetrachloride.

Discussion

Pomace represents approximately 20–35% of the original fruits and is generally composed of remaining carbohydrates, dietary fibers, and small amount of proteins.²⁷ Studies have shown that fruit pomace is a rich source of polyphenolic compounds, therefore making it a good source of natural antioxidants.^12,28 Candrawinata et al. (2014) reported 1148 μg/g phenolics in fresh apple pomace extracted with water.²⁹ So, owing to the significant yield of phenolics in water extract, it can be used as efficient solvent for the extraction of polyphenols. The present investigation was done to study the efficacy of polyphenols-rich industrial APE against CCl₄-induced hepatotoxicity. CCl₄ is metabolized by cytochrome P₄₅₀ into free radicals, which then leads to membrane damage along with the release of inflammatory markers. CYP2E1 plays important role in CCl₄-induced hepatic injury by producing ROS species, thereby leading to apoptosis via oxidative stress.³⁰ The inhibition of CYP2E1 has been proved to be effective against CCl₄-induced liver damage. Earlier tea polyphenols also reported to show the hepatoprotective effect by suppressing the CYP2E1 activity.³¹ So in the current work, in addition to earlier reported CYP2E1 inhibition by polyphenols, we focused to find out other antioxidant mechanism underlying to the hepatoprotective effect of APE.

In concordance with previous studies, present work also indicated that the levels of marker hepatic enzymes ALT, AST, and ALP in serum were significantly increased in CCl₄-treated mice.^17,32 Higher levels of marker enzymes in serum are indicative of membrane damage and their release in the blood.³³ However, the APE administration at doses of 200 and 400 mg/kg significantly reduced the effect of CCl₄ on hepatic damage, thus restoring the normal physiology of the liver and membrane-stabilizing activity. These results were supported by Nie et al., where Red Fuji apple peel and flesh exhibited its protective effect on serum ALT, AST, and ALP levels altered by CCl₄-induced hepatic damage.³⁴ The liver injury markers indicated the dose-dependent protective effect of APE.

SOD is an oxidoreductase enzyme, which helps in the removal of superoxide anion into molecular oxygen and H₂O₂, therefore functions as important antioxidant defense enzyme.³⁵ The decrease in the antioxidative activity of SOD with CCl₄ administration in the present study is consent with earlier reports. Treatment with APE for 14 days at varying dose (200 and 400 mg/kg BW) reversed these changes remarkably. GSH is endogenous antioxidant required for catalysis of electrophilic compounds, thus responsible for xenobiotic, peroxide detoxification in liver. Our study showed decrease levels of redGSH in CCl₄ group, which might be due to the loss of membrane integrity and lipid peroxidation.³⁶ In animals, treated with APE, the serum level of redGSH was increased significantly (p < 0.05) in comparison with the vehicle control group.

Lipid peroxidation in the presence of oxygen results in the production of ROS, which leading to cell membrane damage and thus increasing the serum MDA. Increased levels of MDA shows damage to plasma membrane of cells and formation of free radicals.³⁷ Polyphenols are known to decelerate oxidative damage. The elevated levels of MDA in CCl₄-treated group shows lack of antioxidative capacity due to hepatic damage, whereas APE treatment at dose 200 and 400 mg/kg reduced the liver damage and subsequently increased the activity of antioxidant enzymes.

Histopathology supported the results of biochemical and antioxidant assays. The control group animals showed normal and unaltered histology. Animals administered with CCl₄ were distinct with liver injury and necrosis, while APE remarkably improved the hepatic damage, thus signifying hepatoprotective effect. It has been reported that prolonged exposure to CCl₄ leads to hepatotoxicity via necrosis and apoptosis.³⁸ TUNEL assay showed increased rate of apoptosis as a result of hepatic injury. Besides ameliorating the liver injury, APE treatment also lowered the apoptosis in the hepatocytes.

The transcription factor Nrf2 is a key regulator of antioxidative response elements-mediated gene expression. The Nrf2/ARE signaling pathway is an endogenous system, which involves the synchronized induction of cytoprotective genes that encode antioxidant and anti-inflammatory proteins. Further, the activation of these detoxification genes prevents the pathogenesis of liver disease.³⁹ The Nrf2 transcription factor was earlier shown to protect from ROS and toxin-induced tissue damage through its capacity to induce the expression of the genes encoding ROS and drug-detoxifying enzymes.⁸ It was observed that expression of Nrf2 was altered in the CCl₄-injured livers, whereas treatment with rutin, safflower, and betanin showed activation and upregulation of Nrf2 expression, proving their protective function.⁴⁰ The Nrf2 activation suggested as a novel strategy against CCl₄-induced liver injury and fibrosis.⁴¹ Curcumin reported to attenuate the dimethylnitrosamine-induced liver injury in rats through Nrf2-mediated induction of heme oxygenase 1.⁴² Similarly naringenin, a naturally occurring citrus flavonone, attenuated CCl₄-induced hepatic inflammation by the activation of an Nrf2-mediated pathway in rats.⁴³ In the present study, the slight increase in the Nrf2 protein expression observed in CCl₄ alone-treated group might be a response to induced oxidative stress.²³ The enhanced nuclear expression of Nrf2 following APE treatment is consistent with the increased activities of antioxidant enzymes seen after APE treatment. Nrf2 under usual conditions is bound to Kelch-like ECH-associated protein 1 (Keap1) which act as its repressor. Upon encounter with ROS Keap1 dissociates from Nrf2 eliciting its activation, which then travel to nucleus to activate AREs, primarily two detoxification enzymes: glutathione S-transferase A2 and NADPH: quinone oxidoreductase (NQO1)⁴⁴ for scavenging of free radicals. Thus, Nrf2 plays important role in cellular defense against ROS and electrophilic stress, as seen in case of CCl₄-mediated injury in the present experiment.

In summary, the study showed the protective effect of APE against CCl₄ damage-induced hepatic injury. Treatment with APE reduced the liver damage caused by CCl₄, indicated by improved serum level of liver marker enzymes, antioxidative capacity, apoptosis inhibition, and Nrf2 induction. In addition, study indicated that industrial apple pomace, which is a waste by-product of industry, can be utilized in a better way for the health and nutritional purposes in the future.

Footnotes

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: We acknowledge Director The Council of Scientific and Industrial Research (CSIR)–Institute of Himalayan Bioresource Technology (IHBT) for his support and CSIR, Government of India, for the financial support to carry out this work (Project no. MLP0039 and MLP0074, IHBT publication no. 3818).

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Antioxidant and hepatoprotective effect of polyphenols from apple pomace extract via apoptosis inhibition and Nrf2 activation in mice

Abstract

Keywords

Introduction

Materials and methods

Chemicals and reagents

Raw materials

Extract preparation

Total polyphenol content

DPPH assay

HPLC quantification of phenolics

Experimental animals

Liver injury model

Liver function assays

Antioxidant assays

Lipid peroxidation

Histopathology

Immunohistochemistry

TUNEL assay

Nrf2 expression

Statistical analysis

Results

Total polyphenol and flavonoid content estimations

Free radical scavenging activity

RP-HPLC quantification of polyphenols

Effect of APE on liver injury markers

Effect of APE on antioxidant parameters

Effect of APE on lipid peroxidation

Effect of APE on histopathology of liver

Effect of APE on apoptosis inhibition

Effect of APE on Nrf2 expression in liver

Discussion

Footnotes

Declaration of Conflicting Interests

Funding

References