The Open Medicinal Chemistry Journal




ISSN: 1874-1045 ― Volume 15, 2021
RESEARCH ARTICLE

In Vitro and In Vivo Evaluation of the Protective Potential of Moringa oleifera Against Dietary Acrylamide-induced Toxicity



Lubna Rifai1, Mariam Mohammad2, Karim Raafat3, Fatima A Saleh2, *
1 Department of Nutrition & Dietetics, Faculty of Health Sciences, Beirut Arab University, Beirut, Lebanon
2 Department of Medical Laboratory Sciences, Faculty of Health Sciences, Beirut Arab University, Beirut, Lebanon
3 Department of Pharmaceutical Sciences, Faculty of Pharmacy, Beirut Arab University, Beirut, Lebanon

Abstract

Background:

A c rylamide (AA) in food is a public health concern that has attracted scientists’ attention worldwide.

Objective:

This study was carried out to investigate the efficiency of Moringa oleifera (M. olifera) leaves in the reduction of AA in French fries in vitro and its hepato-protective properties against AA-induced liver toxicity in vivo.

Materials and Methods:

Total phenolic, flavonoid, tannin contents and antioxidant potential of M. oleifera leaves were evaluated and the phenolic constituents characterized via HPLC. AA content was also monitored in French fries using LC-MS/MS. For in vivo assay, mice were treated with AA alone or in combination with M. oleifera (150 and 250 mg/kg IP).

Results:

Phytochemical screening showed that gallic acid, ellagic acid, epicatechin, and quercetin were the most abundant phenolic compounds identified. This work also demonstrated a nearly 37% reduction in AA when French fries were soaked in 1% M. oleifera. Moreover, AA-intoxicated mice resulted in a significant (P < 0.05) elevation in the liver enzyme alanine aminotransferase (ALT), which was restored when pre-treated with M. oleifera.

Conclusion:

This study proved that M. olifera could be effective in reducing AA levels in French fries and that treatment with M. oleifera extract can restore the hepatic damage in AA-intoxicated mice.

Keywords: Moringa oleifera, Acrylamide toxicity, Antioxidant, French fries, Hepato-protective.


Article Information


Identifiers and Pagination:

Year: 2020
Volume: 14
First Page: 26
Last Page: 34
Publisher Id: TOMCJ-14-26
DOI: 10.2174/1874104502014010026

Article History:

Received Date: 24/2/2020
Revision Received Date: 11/5/2020
Acceptance Date: 12/5/2020
Electronic publication date: 30/07/2020
Collection year: 2020

© 2020 Rifai et al.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.


* Address corresponding to this author at the Department of Medical Laboratory Technology, Faculty of Health Sciences, Beirut Arab University, P.O. Box: 11 5020 Beirut, Lebanon; Tel: +961 1 300110 Ext: 2747; E-mail: f.saleh@bau.edu.lb





1. INTRODUCTION

For many years, people have been using heat to cook their food. Heating of food is important to ensure microbiological safety, nutritional quality and the desired sensory properties [1Tucker, G.; Featherstone, S. Essentials of thermal processing., 2011, ]. However, heating could cause the generation of less favourable substances, so-called thermal process contaminants [2Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Törnqvist, M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem., 2002, 50(17), 4998-5006.
[http://dx.doi.org/10.1021/jf020302f] [PMID: 12166997]
, 3Mogol, B.A.; Gökmen, V. Thermal process contaminants: Acrylamide, chloropropanols and furan. Curr. Opin. Food Sci., 2016, 7, 86-92.
[http://dx.doi.org/10.1016/j.cofs.2016.01.005]
]. One such compound that has received much scientific interest in recent years is acrylamide (AA) [4Hamzalıoğlu, A.; Mogol, B.A.; Gökmen, V. Acrylamide: An Overview of the Chemistry and Occurrence in Foods., 2019, , 492-499.]. AA is formed in food products derived from raw materials that are rich in carbohydrates and low in proteins, such as potatoes, when thermally processed at high temperatures (>120°C) [5Ahn, J.S.; Castle, L.; Clarke, D.B.; Lloyd, A.S.; Philo, M.R.; Speck, D.R. Verification of the findings of acrylamide in heated foods. Food Addit. Contam., 2002, 19(12), 1116-1124.
[http://dx.doi.org/10.1080/0265203021000048214] [PMID: 12623671]
, 6Ilona, K.F.; Adriana, N.; Felicia, D.; Sebastian, N.; Manuela, S. Acrylamide, The Healthy Enemy from Food Products that are processed at High Temperatures. Analele Universităţii din Oradea, Fascicula: Ecotoxicologie, Zootehnie şi Tehnologii de Industrie Alimntară., 2015, , 157-164.]. Potato (Solanum tuberosum) is a major agricultural crop consumed daily by millions of people around the world [7Pedreschi, F.; Kaack, K.; Granby, K. Acrylamide content and color development in fried potato strips. Food Res. Int., 2006, 39(1), 40-46.
[http://dx.doi.org/10.1016/j.foodres.2005.06.001]
]. During frying, AA is formed as a result of the Maillard reaction, which is a non-enzymatic browning reaction between asparagines and reducing sugars found in potatoes [8Pedreschi, F.; Kaack, K.; Granby, K. Reduction of acrylamide formation in potato slices during frying. Lebensm. Wiss. Technol., 2004, 37(6), 679-685.
[http://dx.doi.org/10.1016/j.lwt.2004.03.001]
].

Several factors affect the formation of AA in foods, including temperature and time of the heating process, the asparagines and reducing sugars contents as well as pH and moisture content [9Jung, M.Y.; Choi, D.S.; Ju, J.W. A novel technique for limitation of acrylamide formation in fried and baked corn chips and in french fries. J. Food Sci., 2003, 68(4), 1287-1290.
[http://dx.doi.org/10.1111/j.1365-2621.2003.tb09641.x]
]. Manipulation of these factors can regulate AA content in food. For instance, a simple measure of pre-soaking potatoes in water before frying can reduce the formation of AA as soaking decreases the glucose content in the potato strips and in turn reduces AA formation [8Pedreschi, F.; Kaack, K.; Granby, K. Reduction of acrylamide formation in potato slices during frying. Lebensm. Wiss. Technol., 2004, 37(6), 679-685.
[http://dx.doi.org/10.1016/j.lwt.2004.03.001]
]. Blanching in hot or warm water was also reported to attenuate AA formation in French fries not only by reducing the sugar levels of the raw potatoes, but also by decreasing the asparagines content [10Pedreschi, F.; Kaack, K.; Granby, K.; Troncoso, E. Acrylamide reduction under different pre-treatments in French fries. J. Food Eng., 2007, 79(4), 1287-1294.
[http://dx.doi.org/10.1016/j.jfoodeng.2006.04.014]
].

Studies have shown that animal exposure to AA causes reproductive toxicity, genotoxicity, neurotoxicity and hepatic damage [11Rifai, L.; Saleh, F.A. A review on acrylamide in food: Occurrence, toxicity, and mitigation strategies. Int. J. Toxicol., 2020, 39(2), 93-102.
[http://dx.doi.org/10.1177/1091581820902405] [PMID: 32013673]
]. However, only neurotoxicity has been reported in human studies from occupational sources [12He, F.S.; Zhang, S.L.; Wang, H.L.; Li, G.; Zhang, Z.M.; Li, F.L.; Dong, X.M.; Hu, F.R. Neurological and electroneuromyographic assessment of the adverse effects of acrylamide on occupationally exposed workers. Scand. J. Work Environ. Health, 1989, 15(2), 125-129.
[http://dx.doi.org/10.5271/sjweh.1878] [PMID: 2528204]
]. Because of the implications of AA-induced toxicities, investigators were interested in finding natural protective compounds that can reduce AA toxic effects. Studies of natural plants have highlighted the valuable content of numerous polyphenols, which can act as protective agents against AA toxicities. Becalski & colleagues (2002) have shown that AA concentrations were reduced in fried potato slices when rosemary herb, which has a powerful antioxidant activity, was added to the frying oil [13Becalski, A.; Lau, B.P.; Lewis, D.; Seaman, S.W.; Seaman, S.W. Acrylamide in foods: occurrence, sources, and modeling. J. Agric. Food Chem., 2003, 51(3), 802-808.
[http://dx.doi.org/10.1021/jf020889y] [PMID: 12537461]
]. Moreover, Zhang and co-authors (2007) demonstrated that more than 70% of AA was reduced in potato crisps and French fries when dipped into bamboo-leaf extracts, rich in antioxidants [14Zhang, Y.; Chen, J.; Zhang, X.; Wu, X.; Zhang, Y. Addition of antioxidant of bamboo leaves (AOB) effectively reduces acrylamide formation in potato crisps and French fries. J. Agric. Food Chem., 2007, 55(2), 523-528.
[http://dx.doi.org/10.1021/jf062568i] [PMID: 17227088]
].

Moringa oleifera (M. oleifera) belonging to the Moringaceae family, also known as “The Miracle Tree”, is a medium-sized tree that grows widely in many tropical and subtropical countries such as Asia, India, Africa, South and Central America [15Stohs, S.J.; Hartman, M.J. Review of the safety and efficacy of Moringa oleifera Phytother. Res., 2015, 29(6), 796-804.
[http://dx.doi.org/10.1002/ptr.5325] [PMID: 25808883]
]. Recently, this plant has been widely used throughout the world as a valuable food source because of its impressive range of medicinal uses and nutritional properties [16Gopalakrishnan, L.; Doriya, K.; Kumar, D.S. Moringa oleifera: A review on nutritive importance and its medicinal application. Food Sci. Hum. Wellness, 2016, 5(2), 49-56.
[http://dx.doi.org/10.1016/j.fshw.2016.04.001]
]. M. oleifera has also been found to exhibit antioxidant activity due to the high concentrations of antioxidants present in its leaves thus preventing oxidative stress related to many diseases [17Anwar, F.; Latif, S.; Ashraf, M.; Gilani, A.H. Moringa oleifera: A food plant with multiple medicinal uses Phytother. Res., 2007, 21(1), 17-25.
[http://dx.doi.org/10.1002/ptr.2023] [PMID: 17089328]
].

Currently, many studies have proved the effectiveness of several natural compounds against the harmful effects of AA; however, there have been no reports on whether M. oleifera can have such a protective effect. Therefore, in light of many health-promoting benefits attributed to the consumption of M. oleifera, the present study focused on evaluating the phytochemical constituents and antioxidant activity of organic M. oleifera leaves and investigating its potential for reducing AA formation in French fries. Sensory analysis was also conducted in a double-blinded manner. Furthermore, the current study sought to demonstrate the protective effect of this plant against AA-induced hepatotoxicity in mice.

2. MATERIALS AND METHODS

2.1. Plant Material and Chemicals

Organic M. oleifera leaf powder was purchased from MicroIngredientsTM (US); whereas, Lebanese potato tubers and sunflower oil were collected from local commercial sources. Aluminum chloride salt (AlCl3), sodium carbonate anhydrous (NaCO3), absolute ethanol (EtOH, ≥ 99.8% purity), primary secondary amine (PSA) and C18 were obtained from Merck chemical company (Germany). Folin-Ciocalteu (FC) phenol reagent, 2,2- diphenyl-picrylhydrazyl (DPPH), sodium nitrite (NaNO2, ≥ 99.0% purity), gallic acid (C7H6O5), catechin reagents (C15H14O6), absolute methanol (MeOH), magnesium sulphate (MgSO4), and sodium chloride (NaCl) were purchased from Sigma-Aldrich (US). Besides, hydrochloric acid (HCL, 12N), sodium hydroxide (NaOH, 1M), acetonitrile (MeCN) and acrylamide (AA, 99%) standard were obtained from Fluka Company (Switzerland).

2.2. M. oleifera Extraction and Dry Matter Content

The dry matter content of the plant was determined by weighing the appropriate amount of the powder and drying it for 24 hours at 105°C. For extraction, the powdered plant leaf material (120 g/360 ml) was macerated with 80% EtOH for 24 hours at 4oC [18Nadeem, M.; Abdullah, M.; Hussain, I.; Inayat, S.; Javid, A.; Zahoor, Y. Antioxidant potential of Moringa oleifera leaf extract for the stabilisation of butter at refrigeration temperature Czech J. Food Sci., 2013, 31, 332-339.
[http://dx.doi.org/10.17221/366/2012-CJFS]
, 19Ogbunugafor, H.; Igwo-Ezikpe, M.; Igwilo, I.; Ozumba, N.; Adenekan, S.; Ugochukwu, C.; Onyekwelu, O.; Ekechi, A. In vitro and In vivo evaluation of antioxidant properties of Moringa oleifera ethanolic leaves extract and effect on serum lipid indices in rat Maced. J. Med. Sci., 2012, 5(4), 397-403.]. The homogenate mixture was then filtered with the filtrate stored in the dark at 4oC prior to phytochemical analysis. For in vivo work, the extract was concentrated in an oven at 40oC for 48 hours. Then, 0.5 g of the obtained powder was dissolved in 12.5 ml normal saline to obtain a working solution (40mg/ml) which was stored at 4oC for further experiments.

2.3. In Vitro Experimental Study

2.3.1. Determination of Total Phenol Content

Total phenol content (TPC) in ethanolic M. oleifera leaf extract was determined by the Folin-Ciocalteau (FC) method using gallic acid as a standard [20Gajula, D.; Verghese, M.; Boateng, J.; Walker, L.T.; Shackelfor, L.; Mentreddy, S.R.; Cedric, S. Determination of total phenolics, flavonoids and antioxidant and chemopreventive potential of basil (Ocimum basilicum L. and Ocimum tenuiflorum L.). Int. J. Cancer Res., 2009, 5(4), 130-143.
[http://dx.doi.org/10.3923/ijcr.2009.130.143]
]. Briefly, 0.2 ml of the extract was mixed with an FC reagent solution. After 5 min of incubation in the dark at room temperature, 0.8 ml of Na2CO3 (75mg/l) was added and then incubated for 10 min at 60°C. The assay mixture was subjected to a colorimetric measurement at 750nm by UV-VIS spectrophotometer (Gold S54T UV-VIS, China). A blank was prepared with distilled water. TPC in the extract was calculated using the equation: TPC= C*V/m (where C is the sample concentration from the calibration curve (mg/ml), V is the volume (ml) of the solvent used for the extraction, and m represents the weight (g) of the dried sample used) and expressed in mg of Gallic acid equivalence /g dry Dry matter (mg GAE/ g DM). The experiment was done in triplicates and the mean value ± standard deviation (SD) was presented.

2.3.2. Determination of Total Flavonoid Content

Total Flavonoid Content (TFC) in the M. oleifera leaf extract was determined using aluminum trichloride colorimetric method with some modifications and reported as catechin equivalents (CE) per gram of dried weight sample (mg CE/g DM) [21Adom, K.K.; Liu, R.H. Antioxidant activity of grains. J. Agric. Food Chem., 2002, 50(21), 6182-6187.
[http://dx.doi.org/10.1021/jf0205099] [PMID: 12358499]
]. This method is based on the formation of a flavonoid-aluminum complex. Briefly, 1 ml of the extract was added into 4 ml of distilled water followed by 0.3 ml of NaNO2. After 5 min, 0.3 ml of AlCl3 was added to the mixture and at the 6th minute, 2 ml NaOH was poured and mixed. Absorbance was then measured at 510 nm by UV-VIS spectrophotometer. A blank was prepared with distilled water. The TFC of the extracts was calculated using the following equation: TFC = C*V/m.

2.3.3. Determination of Tannin Concentration

Total tannin content (g/l) was determined according to Ribérau-Gayonand and collaborators [22Ribéreau-Gayon, P (1930-2011)., Dubourdieu D (1949-2016)., Lonvaud A (1947-. . . . )., Donèche B (1950-. . . . )., Darriet P (1964-. . . )., Ballestra P, et al. Traité d’oenologie. Tome 1, Microbiologie du vin, vinifications., 1964.]. Total tannin assay is based on the heating process of tannins in an acidic medium leading to the formation of cyanidins. Two tubes were prepared, each containing 1 ml of the M. Oleifera extract sample, 0.5 ml of water and 1.5 ml of 12 N HCl. The first tube was mixed and heated in a water bath at 100°C for 30 min while the second was kept at room temperature for the same duration. Following rapid cooling, 0.25 ml of ethanol was added to the mixture and the resulting absorbance was recorded at 520 nm.

2.3.4. Evaluation of Radical Scavenging Activity

The DPPH radical was used in the current study to screen the radical scavenging activity of M. oleifera extract [23Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol., 1995, 28(1), 25-30.
[http://dx.doi.org/10.1016/S0023-6438(95)80008-5]
]. In short, 4 ml of 1 mM methanolic DPPH solution was added to 0.2 ml of M. oleifera leaf extract. The colorimetric changes from deep-violet to light-yellow after DPPH reduction can be quantified by the decrease in absorbance at 517 nm, after being incubated at room temperature for 30 min in darkness. Methanol was used as the blank. Absorbance of the DPPH radical without antioxidant (the control) was measured. The radical scavenging ability of the sample to scavenge the DPPH radical was evaluated using DPPH scavenging effect (inhibition %) = (Ac−As)×100/Ac, where Ac is the absorbance of the control and As is the absorbance of the sample.

2.3.5. High-Performance Liquid Chromatography with Diode Array Detection (HPLC-DAD) Analysis

Chromatographic analyses were carried out using a mobile phase that consisted of 1% formic acid in double-distilled water (57%) and methanol (43%). The flow rate was 0.7 ml/min at 40°C.

2.3.6. Evaluation of Acrylamide Content in French Fries

2.3.6.1. Sample Preparation

Potato tubers were washed, hand-peeled and cut into strips (1cm x 1cm x 3cm) with a French fries-shaped cutter and rinsed again for 1 min to eliminate starch material adhering to the surface before frying. Raw potato strips were then randomly allocated into separate groups and pre-treated with different solutions to investigate their impact on AA formation. A previously weighed-out 100-gram of potato strips were soaked for 30 min in tap water alone (1L) and tap water with added 1% and 5% M. oleifera leaf powder. A control sample (without any pre-treatment) was also included. Potato strips from each group were then drained and placed separately into a frying basket of an electrical fryer machine and submerged into hot sunflower oil (2.5 L) at 180 ± 5°C for exactly 4.5 min. The fries were then drained for 15 seconds, blotted between two layers of paper towels and cooled at room temperature for about 30 min. Finally, the samples were dried in an oven at 50oC for 48 hours and blended down by an electrical blender until a fine powder was produced. The homogenized powdered French fries were then stored at – 20oC for further analysis.

2.3.6.2. Acrylamide Quantification by LC-MS/MS

AA contents (µg/kg) in potato samples were extracted using QuEChERS method [24Mastovska, K.; Lehotay, S.J. Rapid sample preparation method for LC-MS/MS or GC-MS analysis of acrylamide in various food matrices. J. Agric. Food Chem., 2006, 54(19), 7001-7008.
[http://dx.doi.org/10.1021/jf061330r] [PMID: 16968055]
]. QuEChERS works by using water and acetonitrile to facilitate AA extraction from French fries. To extract AA, 10 ml of water was added to 5 g of thoroughly homogenized samples, vortexed briefly followed by the addition of 10 ml of MeCN. Then 4 g of anhydrous MgSO4 and 1 g of NaCl were added. Immediately after, the tubes were sealed and shaken vigorously for 1 min to prevent the formation of crystalline agglomerates and to ensure sufficient solvent interaction with the entire sample followed by centrifugation for 10 min at 5000 rpm. 1 ml of the MeCN extract was then transferred to a microcentrifuge tube containing 150 mg of PSA, 50 mg end-capped C18 and 150 mg of anhydrous MgSO4 and mixed for 30 seconds. The dispersive solid-phase extraction (SPE) step using PSA is crucial in order to remove any residual carbohydrates or proteins that may interfere with the chromatography analysis. PSA and C18 eliminate the need for hexane defatting and result in clean extracts [25Zhang, Y.; Ren, Y.; Zhang, Y. New research developments on acrylamide: analytical chemistry, formation mechanism, and mitigation recipes. Chem. Rev., 2009, 109(9), 4375-4397.
[http://dx.doi.org/10.1021/cr800318s] [PMID: 19663380]
]. Finally, the mixture was centrifuged for 1 min at 5000 rpm and 500 µl of the supernatant was transferred into an autosampler vial for LC-MS/MS analysis.

2.3.7. Sensory Analysis of French Fries

10 trained panelists evaluated samples of French fries using a 9-point hedonic scale, which typically rates food from ‘dislike extremely’ to ‘like extremely’.

2.4. In Vivo Experimental Study

2.4.1. Experimental Animals

This study was carried out using Male Swiss-Webster mice of 12-16 weeks with initial weight 22-30g, provided by the animal house of Beirut Arab University (BAU) and kept in stainless steel cages under standard laboratory conditions of uniform humidity (50-60%) and temperature (21-23oC) with 12/12 hour light/dark cycle. They were allowed free access to water and standard laboratory pellet diet.

2.4.2. Experimental Design

Mice were randomly divided into 4 groups of 4 animals each and were treated with the particular treatment under consideration as follows; Group I is the control (CTRL) group, Group II is the acrylamide only (AA) group, Group III is the

Table 1
In vivo experimental design.


Fig. (1)
Timeline of in vivo experiments.


AA plus low dose M. oleifera (AA+MLlow) group and group IV is the AA plus high dose M. oleifera (AA+MHhigh) group (Table 1). The M. oleifera extracts at low and high doses (150 and 250 mg/kg, IP) were administered daily for twelve days to Group III and IV respectively, whereas, AA (50 mg/kg IP) was administered on the 13th day for 4 consecutive days as shown in Fig. (1) concomitantly with M. Olifera injections. The AA treated group (Group II) received normal saline in place of M. oleifera extract and the CTRL group received saline only, with the same frequency and duration of treatment of the other groups (Table 1). The injections were given at the same time daily and animal body weights were monitored. At the end of the experiment, animals were sacrificed and serum was collected for subsequent investigation.

2.4.3. Liver Function Test

Activities of serum Alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured spectrophotometrically according to the manufacturer’s instructions using diagnostic kits (Spinreact, Spain). Results were expressed as U/L.

2.5. Statistical Analysis

SPSS version 24 (IBM Co., Armonk, NY, USA) was used for the statistical analysis. For sensory analysis and acrylamide content in French fries, results were analyzed by one-way analysis of variance (ANOVA). Weight and liver test data were analyzed using two way and one way ANOVA, respectively. The analysis was followed by a Post hoc test to compare various groups with each other. Results were expressed as mean ± SD. Differences were considered statistically significant when P < 0.05. The Bonferroni correction was used to adjust confidence intervals.

3. RESULTS

3.1. Phytochemical Analysis of M. Oleifera Leaf Extract

M. oleifera leaf extract was subjected to phytochemical analysis for TPC, TFC and tannins as well as antioxidant activity by DPPH radical scavenging method. TPC determined following a modified Folin-Ciocalteu method was 519.87 ± 61.32 mg GAE/g dry matter. The amount of flavonoids measured was 950 ± 45 mg CE/g dry matter; whereas, the percentage of total tannin content of the M. oleifera leaf extract was 115.78 ± 4.10 mg/l as shown in Table 2 M. Oleifera also exhibited radical scavenging activity of 74.14 %.

3.2. HPLC Profile of M. Oleifera Leaf Ethanol Extract

Phenolic compounds in M. Oleifera ethanol extract were identified and quantified by HPLC as shown in Table 3. HPLC analysis showed the presence of phenolic compounds such as (1) gallic acid (30.7%), (2) catechin (4.13%), (3) chlorogenic acid (2.0%), (4) ellagic acid (30.6%), (5) epi-catechin (9.5%), (6) rutin (5.1%), (7) quercitrin (1.9%), (8) iso-quercitrin (4.8%), (9) quercetin (5.3%), and (10) Kaempferol (4.3%).

Table 2
Total phenolic, flavonoid, tannin content and antioxidant activity of M. oleifera leaf extract.


Table 3
HPLC major phenolic compounds identified in M. oleifera ethanolic extract.


3.3. Acrylamide Content in French Fries

Analysis of AA content in French fries showed that soaking potato strips in water reduced AA formation; however, the reduction was not significant (P > 0.05) compared to the control group which is fried immediately. Interestingly, the addition of M. oleifera to the soaking water reduced AA formation; however the reduction was only significant (P < 0.05) with the application of 1% M. Oleifera (Fig. 2).

3.4. Sensory Analysis

The sensory panel evaluated French fries based on several descriptors linked to appearance, flavor, texture and overall acceptability. The effects of different pre-treatments (1% Vitamin C, 1% M. oleifera and 5% M. oleifera) on sensory properties of French fries are presented in Fig. (3). Potatoes soaked in vitamin C had significant differences in flavor and overall acceptability compared to the control groups (P= 0.001), and 1% M. oleifera (P = 0.002) and 5% M. oleifera (P= 0.011) treated groups. However, the differences in appearance and texture were not significant (P > 0.05) compared to control and M. oleifera treated groups. The appearance, texture, flavor and overall acceptability of the French fries soaked in M. oleifera solutions were not significantly different compared with the control group (P > 0.05) (Fig. 3).

Fig. (2)
Effect of various pre-treatments on acrylamide content in French fries *P < 0.05 indicates significance compared to control samples.


Fig. (3)
Spider web chart of sensory analysis of different treatments on French fries. FI: Fried immediately, MO: M. Oleifera. *P < 0.05 indicates significance compared to control samples.


3.5. Effect of M. oleifera Leaf Extract on Acrylamide-intoxicated Mice

3.5.1. Bodyweight Changes

Fig. (4) presents bodyweight changes in mice groups as per response to different treatments. Mice injected with AA demonstrated loss in bodyweight compared to control untreated mice. Nevertheless, the administration of high dose M. oleifera (250 mg/kg) illustrated a significant increase (P < 0.05) in bodyweight compared to control mice (Fig. 4).

Fig. (4)
Effect of M. oleifera ethanolic leaf extract on body weight in acrylamide-intoxicated mice Data were analyzed by two-way ANOVA following Bonferroni post hoc test. Values were presented as Mean ± SD, n=4. *P < 0.05 indicates significance compared to control samples.


Table 4
Effect of M. oleifera extract on ALT and AST levels in AA intoxicated mice.


3.5.2. Liver Function Test

As shown in Table 4, AA administration significantly (P < 0.05) elevated serum ALT enzyme activity, which indicates liver damage. However, pre-treatment of mice with low and high doses of M. oleifera improved liver function enzymes, with significant (P < 0.05) reduction in ALT activity compared to control.

4. DISCUSSION

Many efforts have been made to reduce AA in the diet and to ameliorate its related toxicity in the body by the use of natural antioxidants. The present study was carried out to evaluate the antiradical activity of M. oleifera and to assess its ability to reduce AA formation in French fries. Additionally, as a powerful phenolic natural agent, the study sought to evaluate the possible protective effect of M. oleifera against AA-induced hepatotoxicity in vivo.

As reported in previous studies, this study demonstrated that M.oleifera is rich in various phytochemicals, mainly gallic and ellagic acid [17Anwar, F.; Latif, S.; Ashraf, M.; Gilani, A.H. Moringa oleifera: A food plant with multiple medicinal uses Phytother. Res., 2007, 21(1), 17-25.
[http://dx.doi.org/10.1002/ptr.2023] [PMID: 17089328]
, 26Bennett, R.N.; Mellon, F.A.; Foidl, N.; Pratt, J.H.; Dupont, M.S.; Perkins, L.; Kroon, P.A. Profiling glucosinolates and phenolics in vegetative and reproductive tissues of the multi-purpose trees Moringa oleifera L. (horseradish tree) and Moringa stenopetala L. J. Agric. Food Chem., 2003, 51(12), 3546-3553.
[http://dx.doi.org/10.1021/jf0211480] [PMID: 12769522]
]. This was in concurrence with other studies showing that gallic acid was among the most abundant phenolics in leaf extract of M. oleifera [27Oboh, G.; Ademiluyi, A.O.; Ademosun, A.O.; Olasehinde, T.A.; Oyeleye, S.I.; Boligon, A.A.; Athayde, M.L. Phenolic extract from moringa oleifera leaves inhibits key enzymes linked to erectile dysfunction and oxidative stress in rats’ penile tissues Biochem. Res. Int., 2015, 2015175950
[http://dx.doi.org/10.1155/2015/175950] [PMID: 26557995]
, 28Alam, P.; Elkholy, S.F.; Mahfouz, S.A.; Alam, P.; Sharaf-Eldin, M.A. HPLC based estimation and extraction of rutin, quercetin and gallic acid in Moringa oleifera plants grown in Saudi Arabia J. Chem. Pharm. Res., 2016, 8(8), 1243-1246.]. M. oleifera also exhibited radical scavenging activity of 74%, which was similar to previously reported result by Falowo et al. (2017), whose investigation showed 75.9% inhibition of DPPH radicals by ethanolic extract of M. oleifera leaves [29Falowo, A.B.; Muchenje, V.; Hugo, A.; Aiyegoro, O.A.; Fayemi, P.O. Antioxidant activities of Moringa oleifera L. and Bidens pilosa L. leaf extracts and their effects on oxidative stability of ground raw beef during refrigeration storage CYTA J. Food, 2017, 15(2), 249-256.
[http://dx.doi.org/10.1080/19476337.2016.1243587]
].

AA concentration in French fries was also tested prior to and after treatment. Our findings revealed that soaking in water for 30 min was able to reduce AA levels in French fries, which was in accordance with other investigators who demonstrated reductions in AA formation when potato slices were soaked in water due to the decrease in the glucose content of the potato strips before frying [8Pedreschi, F.; Kaack, K.; Granby, K. Reduction of acrylamide formation in potato slices during frying. Lebensm. Wiss. Technol., 2004, 37(6), 679-685.
[http://dx.doi.org/10.1016/j.lwt.2004.03.001]
, 9Jung, M.Y.; Choi, D.S.; Ju, J.W. A novel technique for limitation of acrylamide formation in fried and baked corn chips and in french fries. J. Food Sci., 2003, 68(4), 1287-1290.
[http://dx.doi.org/10.1111/j.1365-2621.2003.tb09641.x]
]. On the other side, soaking potato strips in water containing M. oleifera (1%), significantly reduced AA formation in French fries when compared to the control group (fried immediately). This can be explained by the fact that M. oleifera is rich in phenolic compounds. Several studies have reported how high levels of phenolic compounds may mitigate AA formation [30Ismial, S.A.; Ali, R.F.; Askar, M.S.; Samy, W.M. Impact of pre-treatments on the acrylamide formation and organoleptic evolution of fried potato chips. Am. J. Biochem. Biotechnol., 2013, 9(2), 90-101.
[http://dx.doi.org/10.3844/ajbbsp.2013.90.101]
]. Ismial and colleagues (2013) demonstrated that soaking potato strips in phenolic solutions like ferulic acid, protocatechoic acid, caffeic acid, catechin and gallic acid for 60 min resulted in significant reductions in AA content in fried potatoes [30Ismial, S.A.; Ali, R.F.; Askar, M.S.; Samy, W.M. Impact of pre-treatments on the acrylamide formation and organoleptic evolution of fried potato chips. Am. J. Biochem. Biotechnol., 2013, 9(2), 90-101.
[http://dx.doi.org/10.3844/ajbbsp.2013.90.101]
]. It is also worth noting that the organoleptic features of French fries were not affected by the M. oleifera treatment.

Besides, the present study determined that AA administration in vivo decreased bodyweight which is in agreement with other findings [31Mahmood, S.A.F.; Amin, K.A.M.; Salih, S.F.M. Effect of acrylamide on liver and kidneys in albino wistar rats. Int. J. Curr. Microbiol. Appl. Sci., 2015, 4(5), 434-444.]. Mahmood et al. (2015) proposed that this decrease in bodyweight could result from the breakdown of tissue and blood cells in the liver and kidney by AA in rats [31Mahmood, S.A.F.; Amin, K.A.M.; Salih, S.F.M. Effect of acrylamide on liver and kidneys in albino wistar rats. Int. J. Curr. Microbiol. Appl. Sci., 2015, 4(5), 434-444.]. However, M. oleifera treatment seems to significantly enhance bodyweight which was in accordance with Adeyemi and collaborators who reported improvements in bodyweight of mice when treated with M. oleifera after Nickel exposure [32Adeyemi, O.S.; Elebiyo, T.C. Moringa oleifera supplemented diets prevented nickel-induced nephrotoxicity in wistar rats J. Nutr. Metab., 2014, 2014958621
[http://dx.doi.org/10.1155/2014/958621] [PMID: 25295181]
]. This increase in the bodyweight of the group pre-treated with M. oleifera indicates its high nutritional values.

In order to confirm the AA-induced liver toxicity, liver enzymes (ALT and AST) activities were determined. AA intoxication showed hepatotoxicity manifested in a significant elevation in serum ALT which is in harmony with other studies [33Kovac, R.; Rajkovic, V.; Koledin, I.; Matavulj, M. Acrylamide alters glycogen content and enzyme activities in the liver of juvenile rat. Acta Histochem., 2015, 117(8), 712-717.
[http://dx.doi.org/10.1016/j.acthis.2015.09.004] [PMID: 26475516]
-35Erdemli, M.; Sahin, N.; Turkoz, Y.; Yilmaz, I.; Cinar, K.; Akgoz, M.; Cigremis, Y. The possible hepatoprotective effect of apricot against acrylamide induced hepatotoxicity in rats. J Turgut Ozal Med Cent., 2017, 24(1), 48-53.
[http://dx.doi.org/10.5455/jtomc.2016.12.125]
]. However, serum AST level was not significantly different from that of the control group. Although both ALT and AST levels are usually elevated when the liver is intoxicated, ALT is more specific in detecting liver damage as a result of drugs or other substances that are toxic to the liver and, in some cases, may be the only one to be significantly increased [36Giannini, E.G.; Testa, R.; Savarino, V. Liver enzyme alteration: a guide for clinicians. CMAJ, 2005, 172(3), 367-379.
[http://dx.doi.org/10.1503/cmaj.1040752] [PMID: 15684121]
-38Mohamed, J.; Nazratun Nafizah, A.H.; Zariyantey, A.H.; Budin, S.B. Mechanisms of diabetes-induced liver damage: The role of oxidative stress and inflammation. Sultan Qaboos Univ. Med. J., 2016, 16(2), e132-e141.
[http://dx.doi.org/10.18295/squmj.2016.16.02.002] [PMID: 27226903]
]. This comes in line with Kim et al. (2008) who reported that the serum ALT activity is a reliable and sensitive marker of liver disease and is more commonly elevated than AST [39Kim, W.R.; Flamm, S.L.; Di Bisceglie, A.M.; Bodenheimer, H.C. Serum activity of alanine aminotransferase (ALT) as an indicator of health and disease. Hepatology, 2008, 47(4), 1363-1370.
[http://dx.doi.org/10.1002/hep.22109] [PMID: 18366115]
]. Furthermore, while increased AST levels are indicative of a tissue injury, it is not specific to the liver [40Borlak, J.; Chougule, A.; Singh, P.K. How useful are clinical liver function tests in in vitro human hepatotoxicity assays? Toxicol. In Vitro, 2014, 28(5), 784-795.
[http://dx.doi.org/10.1016/j.tiv.2014.03.006] [PMID: 24685772]
]. On contrary, any elevation of ALT is a direct indication of a liver injury [40Borlak, J.; Chougule, A.; Singh, P.K. How useful are clinical liver function tests in in vitro human hepatotoxicity assays? Toxicol. In Vitro, 2014, 28(5), 784-795.
[http://dx.doi.org/10.1016/j.tiv.2014.03.006] [PMID: 24685772]
]. Pretreatments of mice with M. oleifera extract were able to afford protection against AA-induced liver damage by significantly lowering the elevated ALT levels. This reduction might be a result of the regulation of liver cell membrane integrity by M. oleifera due to its antioxidant and free radical scavenging properties [32Adeyemi, O.S.; Elebiyo, T.C. Moringa oleifera supplemented diets prevented nickel-induced nephrotoxicity in wistar rats J. Nutr. Metab., 2014, 2014958621
[http://dx.doi.org/10.1155/2014/958621] [PMID: 25295181]
]. Herein, there is a clear demonstration of the hepato-protective effects of M. oleifera against Acrylamide-induced toxicity.

CONCLUSION

To the best of our knowledge, this study is the first to investigate the potential of M. oleifera in reducing AA formation in French fries as well as its hepato-protective effect against AA toxicity in vivo . Our findings show that the addition of M. oleifera presents an attractive and promising strategy to reduce AA formation in potato-based products while also retaining reasonable sensory attributes. M. oleifera can also be useful as a liver-protective natural agent especially when added to starch-containing foods before frying. The approach described here could be considered as a pioneer finding of a simple and natural way to reduce the amounts of AA in potato-based foods and its toxic effects, and thus can be an attractive candidate for future implementation in the food industry.

ETHICS APPROVAL AND CONSENT TO PARTICIPATE

All animals were treated according to ethical standards and the guidelines of the Ministry of Higher Education and Legislation of animal experiments with the approval of BAU-Institutional Review Board, Beirut, Lebanon (2019H-0055-HS-R-0310).

HUMAN AND ANIMAL RIGHTS

Human participants were not included in this study. All animal procedures were carried out while abiding by the US National Research Council's “Guide for the Care and Use of Laboratory Animals”.

CONSENT FOR PUBLICATION

Not applicable.

AVAILABILITY OF DATA AND MATERIALS

The authors confirm that the data supporting the findings of this research are available within the article.

FUNDING

None.

CONFLICT OF INTEREST

The authors declare no conflict of interest, financial or otherwise.

ACKNOWLEDGEMENTS

I would like to thank Mrs. Lama Hanbali for laboratory assistance.

REFERENCES

[1] Tucker, G.; Featherstone, S. Essentials of thermal processing., 2011,
[2] Tareke, E.; Rydberg, P.; Karlsson, P.; Eriksson, S.; Törnqvist, M. Analysis of acrylamide, a carcinogen formed in heated foodstuffs. J. Agric. Food Chem., 2002, 50(17), 4998-5006.
[http://dx.doi.org/10.1021/jf020302f] [PMID: 12166997]
[3] Mogol, B.A.; Gökmen, V. Thermal process contaminants: Acrylamide, chloropropanols and furan. Curr. Opin. Food Sci., 2016, 7, 86-92.
[http://dx.doi.org/10.1016/j.cofs.2016.01.005]
[4] Hamzalıoğlu, A.; Mogol, B.A.; Gökmen, V. Acrylamide: An Overview of the Chemistry and Occurrence in Foods., 2019, , 492-499.
[5] Ahn, J.S.; Castle, L.; Clarke, D.B.; Lloyd, A.S.; Philo, M.R.; Speck, D.R. Verification of the findings of acrylamide in heated foods. Food Addit. Contam., 2002, 19(12), 1116-1124.
[http://dx.doi.org/10.1080/0265203021000048214] [PMID: 12623671]
[6] Ilona, K.F.; Adriana, N.; Felicia, D.; Sebastian, N.; Manuela, S. Acrylamide, The Healthy Enemy from Food Products that are processed at High Temperatures. Analele Universităţii din Oradea, Fascicula: Ecotoxicologie, Zootehnie şi Tehnologii de Industrie Alimntară., 2015, , 157-164.
[7] Pedreschi, F.; Kaack, K.; Granby, K. Acrylamide content and color development in fried potato strips. Food Res. Int., 2006, 39(1), 40-46.
[http://dx.doi.org/10.1016/j.foodres.2005.06.001]
[8] Pedreschi, F.; Kaack, K.; Granby, K. Reduction of acrylamide formation in potato slices during frying. Lebensm. Wiss. Technol., 2004, 37(6), 679-685.
[http://dx.doi.org/10.1016/j.lwt.2004.03.001]
[9] Jung, M.Y.; Choi, D.S.; Ju, J.W. A novel technique for limitation of acrylamide formation in fried and baked corn chips and in french fries. J. Food Sci., 2003, 68(4), 1287-1290.
[http://dx.doi.org/10.1111/j.1365-2621.2003.tb09641.x]
[10] Pedreschi, F.; Kaack, K.; Granby, K.; Troncoso, E. Acrylamide reduction under different pre-treatments in French fries. J. Food Eng., 2007, 79(4), 1287-1294.
[http://dx.doi.org/10.1016/j.jfoodeng.2006.04.014]
[11] Rifai, L.; Saleh, F.A. A review on acrylamide in food: Occurrence, toxicity, and mitigation strategies. Int. J. Toxicol., 2020, 39(2), 93-102.
[http://dx.doi.org/10.1177/1091581820902405] [PMID: 32013673]
[12] He, F.S.; Zhang, S.L.; Wang, H.L.; Li, G.; Zhang, Z.M.; Li, F.L.; Dong, X.M.; Hu, F.R. Neurological and electroneuromyographic assessment of the adverse effects of acrylamide on occupationally exposed workers. Scand. J. Work Environ. Health, 1989, 15(2), 125-129.
[http://dx.doi.org/10.5271/sjweh.1878] [PMID: 2528204]
[13] Becalski, A.; Lau, B.P.; Lewis, D.; Seaman, S.W.; Seaman, S.W. Acrylamide in foods: occurrence, sources, and modeling. J. Agric. Food Chem., 2003, 51(3), 802-808.
[http://dx.doi.org/10.1021/jf020889y] [PMID: 12537461]
[14] Zhang, Y.; Chen, J.; Zhang, X.; Wu, X.; Zhang, Y. Addition of antioxidant of bamboo leaves (AOB) effectively reduces acrylamide formation in potato crisps and French fries. J. Agric. Food Chem., 2007, 55(2), 523-528.
[http://dx.doi.org/10.1021/jf062568i] [PMID: 17227088]
[15] Stohs, S.J.; Hartman, M.J. Review of the safety and efficacy of Moringa oleifera Phytother. Res., 2015, 29(6), 796-804.
[http://dx.doi.org/10.1002/ptr.5325] [PMID: 25808883]
[16] Gopalakrishnan, L.; Doriya, K.; Kumar, D.S. Moringa oleifera: A review on nutritive importance and its medicinal application. Food Sci. Hum. Wellness, 2016, 5(2), 49-56.
[http://dx.doi.org/10.1016/j.fshw.2016.04.001]
[17] Anwar, F.; Latif, S.; Ashraf, M.; Gilani, A.H. Moringa oleifera: A food plant with multiple medicinal uses Phytother. Res., 2007, 21(1), 17-25.
[http://dx.doi.org/10.1002/ptr.2023] [PMID: 17089328]
[18] Nadeem, M.; Abdullah, M.; Hussain, I.; Inayat, S.; Javid, A.; Zahoor, Y. Antioxidant potential of Moringa oleifera leaf extract for the stabilisation of butter at refrigeration temperature Czech J. Food Sci., 2013, 31, 332-339.
[http://dx.doi.org/10.17221/366/2012-CJFS]
[19] Ogbunugafor, H.; Igwo-Ezikpe, M.; Igwilo, I.; Ozumba, N.; Adenekan, S.; Ugochukwu, C.; Onyekwelu, O.; Ekechi, A. In vitro and In vivo evaluation of antioxidant properties of Moringa oleifera ethanolic leaves extract and effect on serum lipid indices in rat Maced. J. Med. Sci., 2012, 5(4), 397-403.
[20] Gajula, D.; Verghese, M.; Boateng, J.; Walker, L.T.; Shackelfor, L.; Mentreddy, S.R.; Cedric, S. Determination of total phenolics, flavonoids and antioxidant and chemopreventive potential of basil (Ocimum basilicum L. and Ocimum tenuiflorum L.). Int. J. Cancer Res., 2009, 5(4), 130-143.
[http://dx.doi.org/10.3923/ijcr.2009.130.143]
[21] Adom, K.K.; Liu, R.H. Antioxidant activity of grains. J. Agric. Food Chem., 2002, 50(21), 6182-6187.
[http://dx.doi.org/10.1021/jf0205099] [PMID: 12358499]
[22] Ribéreau-Gayon, P (1930-2011)., Dubourdieu D (1949-2016)., Lonvaud A (1947-. . . . )., Donèche B (1950-. . . . )., Darriet P (1964-. . . )., Ballestra P, et al. Traité d’oenologie. Tome 1, Microbiologie du vin, vinifications., 1964.
[23] Brand-Williams, W.; Cuvelier, M.E.; Berset, C. Use of a free radical method to evaluate antioxidant activity. Lebensm. Wiss. Technol., 1995, 28(1), 25-30.
[http://dx.doi.org/10.1016/S0023-6438(95)80008-5]
[24] Mastovska, K.; Lehotay, S.J. Rapid sample preparation method for LC-MS/MS or GC-MS analysis of acrylamide in various food matrices. J. Agric. Food Chem., 2006, 54(19), 7001-7008.
[http://dx.doi.org/10.1021/jf061330r] [PMID: 16968055]
[25] Zhang, Y.; Ren, Y.; Zhang, Y. New research developments on acrylamide: analytical chemistry, formation mechanism, and mitigation recipes. Chem. Rev., 2009, 109(9), 4375-4397.
[http://dx.doi.org/10.1021/cr800318s] [PMID: 19663380]
[26] Bennett, R.N.; Mellon, F.A.; Foidl, N.; Pratt, J.H.; Dupont, M.S.; Perkins, L.; Kroon, P.A. Profiling glucosinolates and phenolics in vegetative and reproductive tissues of the multi-purpose trees Moringa oleifera L. (horseradish tree) and Moringa stenopetala L. J. Agric. Food Chem., 2003, 51(12), 3546-3553.
[http://dx.doi.org/10.1021/jf0211480] [PMID: 12769522]
[27] Oboh, G.; Ademiluyi, A.O.; Ademosun, A.O.; Olasehinde, T.A.; Oyeleye, S.I.; Boligon, A.A.; Athayde, M.L. Phenolic extract from moringa oleifera leaves inhibits key enzymes linked to erectile dysfunction and oxidative stress in rats’ penile tissues Biochem. Res. Int., 2015, 2015175950
[http://dx.doi.org/10.1155/2015/175950] [PMID: 26557995]
[28] Alam, P.; Elkholy, S.F.; Mahfouz, S.A.; Alam, P.; Sharaf-Eldin, M.A. HPLC based estimation and extraction of rutin, quercetin and gallic acid in Moringa oleifera plants grown in Saudi Arabia J. Chem. Pharm. Res., 2016, 8(8), 1243-1246.
[29] Falowo, A.B.; Muchenje, V.; Hugo, A.; Aiyegoro, O.A.; Fayemi, P.O. Antioxidant activities of Moringa oleifera L. and Bidens pilosa L. leaf extracts and their effects on oxidative stability of ground raw beef during refrigeration storage CYTA J. Food, 2017, 15(2), 249-256.
[http://dx.doi.org/10.1080/19476337.2016.1243587]
[30] Ismial, S.A.; Ali, R.F.; Askar, M.S.; Samy, W.M. Impact of pre-treatments on the acrylamide formation and organoleptic evolution of fried potato chips. Am. J. Biochem. Biotechnol., 2013, 9(2), 90-101.
[http://dx.doi.org/10.3844/ajbbsp.2013.90.101]
[31] Mahmood, S.A.F.; Amin, K.A.M.; Salih, S.F.M. Effect of acrylamide on liver and kidneys in albino wistar rats. Int. J. Curr. Microbiol. Appl. Sci., 2015, 4(5), 434-444.
[32] Adeyemi, O.S.; Elebiyo, T.C. Moringa oleifera supplemented diets prevented nickel-induced nephrotoxicity in wistar rats J. Nutr. Metab., 2014, 2014958621
[http://dx.doi.org/10.1155/2014/958621] [PMID: 25295181]
[33] Kovac, R.; Rajkovic, V.; Koledin, I.; Matavulj, M. Acrylamide alters glycogen content and enzyme activities in the liver of juvenile rat. Acta Histochem., 2015, 117(8), 712-717.
[http://dx.doi.org/10.1016/j.acthis.2015.09.004] [PMID: 26475516]
[34] Al-Qahtani, F.A.; Arafah, M.; Sharma, B.; Siddiqi, N.J. Effects of alpha lipoic acid on acrylamide-induced hepatotoxicity in rats. Cell. Mol. Biol., 2017, 63(6), 1-6.
[http://dx.doi.org/10.14715/cmb/2017.63.6.1] [PMID: 28968200]
[35] Erdemli, M.; Sahin, N.; Turkoz, Y.; Yilmaz, I.; Cinar, K.; Akgoz, M.; Cigremis, Y. The possible hepatoprotective effect of apricot against acrylamide induced hepatotoxicity in rats. J Turgut Ozal Med Cent., 2017, 24(1), 48-53.
[http://dx.doi.org/10.5455/jtomc.2016.12.125]
[36] Giannini, E.G.; Testa, R.; Savarino, V. Liver enzyme alteration: a guide for clinicians. CMAJ, 2005, 172(3), 367-379.
[http://dx.doi.org/10.1503/cmaj.1040752] [PMID: 15684121]
[37] Gowda, S.; Desai, P.B.; Hull, V.V.; Math, A.A.K.; Vernekar, S.N.; Kulkarni, S.S. A review on laboratory liver function tests. Pan Afr. Med. J., 2009, 3, 17.
[PMID: 21532726]
[38] Mohamed, J.; Nazratun Nafizah, A.H.; Zariyantey, A.H.; Budin, S.B. Mechanisms of diabetes-induced liver damage: The role of oxidative stress and inflammation. Sultan Qaboos Univ. Med. J., 2016, 16(2), e132-e141.
[http://dx.doi.org/10.18295/squmj.2016.16.02.002] [PMID: 27226903]
[39] Kim, W.R.; Flamm, S.L.; Di Bisceglie, A.M.; Bodenheimer, H.C. Serum activity of alanine aminotransferase (ALT) as an indicator of health and disease. Hepatology, 2008, 47(4), 1363-1370.
[http://dx.doi.org/10.1002/hep.22109] [PMID: 18366115]
[40] Borlak, J.; Chougule, A.; Singh, P.K. How useful are clinical liver function tests in in vitro human hepatotoxicity assays? Toxicol. In Vitro, 2014, 28(5), 784-795.
[http://dx.doi.org/10.1016/j.tiv.2014.03.006] [PMID: 24685772]
Track Your Manuscript:


Endorsements



"Open access will revolutionize 21st century knowledge work and accelerate the diffusion of ideas and evidence that support just in time learning and the evolution of thinking in a number of disciplines."


Daniel Pesut
(Indiana University School of Nursing, USA)

"It is important that students and researchers from all over the world can have easy access to relevant, high-standard and timely scientific information. This is exactly what Open Access Journals provide and this is the reason why I support this endeavor."


Jacques Descotes
(Centre Antipoison-Centre de Pharmacovigilance, France)

"Publishing research articles is the key for future scientific progress. Open Access publishing is therefore of utmost importance for wider dissemination of information, and will help serving the best interest of the scientific community."


Patrice Talaga
(UCB S.A., Belgium)

"Open access journals are a novel concept in the medical literature. They offer accessible information to a wide variety of individuals, including physicians, medical students, clinical investigators, and the general public. They are an outstanding source of medical and scientific information."


Jeffrey M. Weinberg
(St. Luke's-Roosevelt Hospital Center, USA)

"Open access journals are extremely useful for graduate students, investigators and all other interested persons to read important scientific articles and subscribe scientific journals. Indeed, the research articles span a wide range of area and of high quality. This is specially a must for researchers belonging to institutions with limited library facility and funding to subscribe scientific journals."


Debomoy K. Lahiri
(Indiana University School of Medicine, USA)

"Open access journals represent a major break-through in publishing. They provide easy access to the latest research on a wide variety of issues. Relevant and timely articles are made available in a fraction of the time taken by more conventional publishers. Articles are of uniformly high quality and written by the world's leading authorities."


Robert Looney
(Naval Postgraduate School, USA)

"Open access journals have transformed the way scientific data is published and disseminated: particularly, whilst ensuring a high quality standard and transparency in the editorial process, they have increased the access to the scientific literature by those researchers that have limited library support or that are working on small budgets."


Richard Reithinger
(Westat, USA)

"Not only do open access journals greatly improve the access to high quality information for scientists in the developing world, it also provides extra exposure for our papers."


J. Ferwerda
(University of Oxford, UK)

"Open Access 'Chemistry' Journals allow the dissemination of knowledge at your finger tips without paying for the scientific content."


Sean L. Kitson
(Almac Sciences, Northern Ireland)

"In principle, all scientific journals should have open access, as should be science itself. Open access journals are very helpful for students, researchers and the general public including people from institutions which do not have library or cannot afford to subscribe scientific journals. The articles are high standard and cover a wide area."


Hubert Wolterbeek
(Delft University of Technology, The Netherlands)

"The widest possible diffusion of information is critical for the advancement of science. In this perspective, open access journals are instrumental in fostering researches and achievements."


Alessandro Laviano
(Sapienza - University of Rome, Italy)

"Open access journals are very useful for all scientists as they can have quick information in the different fields of science."


Philippe Hernigou
(Paris University, France)

"There are many scientists who can not afford the rather expensive subscriptions to scientific journals. Open access journals offer a good alternative for free access to good quality scientific information."


Fidel Toldrá
(Instituto de Agroquimica y Tecnologia de Alimentos, Spain)

"Open access journals have become a fundamental tool for students, researchers, patients and the general public. Many people from institutions which do not have library or cannot afford to subscribe scientific journals benefit of them on a daily basis. The articles are among the best and cover most scientific areas."


M. Bendandi
(University Clinic of Navarre, Spain)

"These journals provide researchers with a platform for rapid, open access scientific communication. The articles are of high quality and broad scope."


Peter Chiba
(University of Vienna, Austria)

"Open access journals are probably one of the most important contributions to promote and diffuse science worldwide."


Jaime Sampaio
(University of Trás-os-Montes e Alto Douro, Portugal)

"Open access journals make up a new and rather revolutionary way to scientific publication. This option opens several quite interesting possibilities to disseminate openly and freely new knowledge and even to facilitate interpersonal communication among scientists."


Eduardo A. Castro
(INIFTA, Argentina)

"Open access journals are freely available online throughout the world, for you to read, download, copy, distribute, and use. The articles published in the open access journals are high quality and cover a wide range of fields."


Kenji Hashimoto
(Chiba University, Japan)

"Open Access journals offer an innovative and efficient way of publication for academics and professionals in a wide range of disciplines. The papers published are of high quality after rigorous peer review and they are Indexed in: major international databases. I read Open Access journals to keep abreast of the recent development in my field of study."


Daniel Shek
(Chinese University of Hong Kong, Hong Kong)

"It is a modern trend for publishers to establish open access journals. Researchers, faculty members, and students will be greatly benefited by the new journals of Bentham Science Publishers Ltd. in this category."


Jih Ru Hwu
(National Central University, Taiwan)


Browse Contents




Webmaster Contact: info@benthamopen.net
Copyright © 2021 Bentham Open